Family of pesticidal proteins and methods for their use

ABSTRACT

Compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are provided. Compositions comprising a coding sequence for pesticidal polypeptides are provided. The coding sequences can be used in DNA constructs or expression cassettes for transformation and expression in plants and bacteria. Compositions also comprise transformed bacteria, plants, plant cells, tissues, and seeds. In particular, isolated pesticidal nucleic acid molecules are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed. In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequence shown in SEQ ID NO:2, 4, 6, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 61, the nucleotide sequence set forth in SEQ ID NO:1, 3, 5, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 60, or the nucleotide sequence deposited in a bacterial host as Accession No. NRRL B-30961, B-30955, B-30956, B-30957, B-30958, B-30942, B-30939, B-30941, B-50047, B-50047, B-30959, B-30960, B-30943, B-50048, or B-50048, as well as variants and fragments thereof.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.Nos. 60/814,007, filed Jun. 15, 2006; 60/813,859, filed Jun. 15, 2006;60/814,420, filed Jun. 16, 2006; 60/814,212, filed Jun. 16, 2006; and60/814,989, filed Jun. 20, 2006, the contents of which are hereinincorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named“329211_SequenceListing.txt”, created on Jun. 14, 2007, and having asize of 305 kilobytes and is filed concurrently with the specification.The sequence listing contained in this ASCII formatted document is partof the specification and is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

This invention relates to the field of molecular biology. Provided arenovel genes that encode pesticidal proteins. These proteins and thenucleic acid sequences that encode them are useful in preparingpesticidal formulations and in the production of transgenicpest-resistant plants.

BACKGROUND OF THE INVENTION

Bacillus thuringiensis is a Gram-positive spore forming soil bacteriumcharacterized by its ability to produce crystalline inclusions that arespecifically toxic to certain orders and species of insects, but areharmless to plants and other non-targeted organisms. For this reason,compositions including Bacillus thuringiensis strains or theirinsecticidal proteins can be used as environmentally-acceptableinsecticides to control agricultural insect pests or insect vectors fora variety of human or animal diseases.

Crystal (Cry) proteins (delta-endotoxins) from Bacillus thuringiensishave potent insecticidal activity against predominantly Lepidopteran,Dipteran, and Coleopteran larvae. These proteins also have shownactivity against Hymenoptera, Homoptera, Phthiraptera, Mallophaga, andAcari pest orders, as well as other invertebrate orders such asNemathelminthes, Platyhelminthes, and Sarcomastigorphora (Feitelson(1993) The Bacillus Thuringiensis family tree. In Advanced EngineeredPesticides, Marcel Dekker, Inc., New York, N.Y.) These proteins wereoriginally classified as CryI to CryV based primarily on theirinsecticidal activity. The major classes were Lepidoptera-specific (I),Lepidoptera- and Diptera-specific (II), Coleoptera-specific (III),Diptera-specific (IV), and nematode-specific (V) and (VI). The proteinswere further classified into subfamilies; more highly related proteinswithin each family were assigned divisional letters such as Cry1A,Cry1B, Cry1C, etc. Even more closely related proteins within eachdivision were given names such as Cry1C1, Cry1C2, etc.

A new nomenclature was recently described for the Cry genes based uponamino acid sequence homology rather than insect target specificity(Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62:807-813). In thenew classification, each toxin is assigned a unique name incorporating aprimary rank (an Arabic number), a secondary rank (an uppercase letter),a tertiary rank (a lowercase letter), and a quaternary rank (anotherArabic number). In the new classification, Roman numerals have beenexchanged for Arabic numerals in the primary rank. Proteins with lessthan 45% sequence identity have different primary ranks, and thecriteria for secondary and tertiary ranks are 78% and 95%, respectively.

The crystal protein does not exhibit insecticidal activity until it hasbeen ingested and solubilized in the insect midgut. The ingestedprotoxin is hydrolyzed by proteases in the insect digestive tract to anactive toxic molecule. (Höfte and Whiteley (1989) Microbiol. Rev.53:242-255). This toxin binds to apical brush border receptors in themidgut of the target larvae and inserts into the apical membranecreating ion channels or pores, resulting in larval death.

Delta-endotoxins generally have five conserved sequence domains, andthree conserved structural domains (see, for example, de Maagd et al.(2001) Trends Genetics 17:193-199). The first conserved structuraldomain consists of seven alpha helices and is involved in membraneinsertion and pore formation. Domain II consists of three beta-sheetsarranged in a Greek key configuration, and domain III consists of twoantiparallel beta-sheets in “jelly-roll” formation (de Maagd et al.,2001, supra). Domains II and III are involved in receptor recognitionand binding, and are therefore considered determinants of toxinspecificity.

Because of the devastation that insects can confer, and the improvementin yield by controlling insect pests, there is a continual need todiscover new forms of pesticidal toxins.

SUMMARY OF INVENTION

Compositions and methods for conferring pesticidal activity to bacteria,plants, plant cells, tissues and seeds are provided. Compositionsinclude nucleic acid molecules encoding sequences for pesticidal andinsectidal polypeptides, vectors comprising those nucleic acidmolecules, and host cells comprising the vectors. Compositions alsoinclude the pesticidal polypeptide sequences and antibodies to thosepolypeptides. The nucleotide sequences can be used in DNA constructs orexpression cassettes for transformation and expression in organisms,including microorganisms and plants. The nucleotide or amino acidsequences may be synthetic sequences that have been designed forexpression in an organism including, but not limited to, a microorganismor a plant. Compositions also comprise transformed bacteria, plants,plant cells, tissues, and seeds.

In particular, isolated nucleic acid molecules are provided that encodea pesticidal protein. Additionally, amino acid sequences correspondingto the pesticidal protein are encompassed. In particular, the presentinvention provides for an isolated nucleic acid molecule comprising anucleotide sequence encoding the amino acid sequence shown in SEQ IDNO:2, 4, 6, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37or 61, a nucleotide sequence set forth in SEQ ID NO:1, 3, 5, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 60, or thedelta-endotoxin nucleotide sequence of the DNA insert of the plasmiddeposited in a bacterial host as Accession No. NRRL B-30961, B-30955,B-30956, B-30957, B-30958, B-30942, B-30939, B-30941, B-50047, B-50047,B-30959, B-30960, B-30943, B-50048, or B-50048, as well as variants andfragments thereof. Nucleotide sequences that are complementary to anucleotide sequence of the invention, or that hybridize to a sequence ofthe invention are also encompassed.

Methods are provided for producing the polypeptides of the invention,and for using those polypeptides for controlling or killing alepidopteran, coleopteran, nematode, or dipteran pest. Methods and kitsfor detecting the nucleic acids and polypeptides of the invention in asample are also included.

The compositions and methods of the invention are useful for theproduction of organisms with enhanced pest resistance or tolerance.These organisms and compositions comprising the organisms are desirablefor agricultural purposes. The compositions of the invention are alsouseful for generating altered or improved proteins that have pesticidalactivity, or for detecting the presence of pesticidal proteins ornucleic acids in products or organisms.

DESCRIPTION OF FIGURES

FIG. 1 shows an alignment of AXMI-022 with the Iota1b from Clostridiumperfringens (SEQ ID NO:49), Isp1A from Brevibacillus laterosporus (SEQID NO:50), Isp1B from Brevibacillus laterosporus (SEQ ID NO:51), Vip1Abfrom Bacillus thuringiensis (SEQ ID NO:52), and Vip1Ac from Bacillusthuringiensis (SEQ ID NO:53). The alignment shows the most highlyconserved amino acid residues highlighted in black, and highly conservedamino acid residues highlighted in gray.

FIG. 2 shows an alignment of AXMI-022 with Vip1Ab (SEQ ID NO:52). Thealignment shows the most highly conserved amino acid residueshighlighted in black, and highly conserved amino acid residueshighlighted in gray.

FIG. 3 shows an alignment of AXMI-022 with Cry 37Aa1 from Bacillusthuringiensis (SEQ ID NO:54). The alignment shows the most highlyconserved amino acid residues highlighted in black, and highly conservedamino acid residues highlighted in gray.

FIG. 4 shows an alignment of AXMI-023 with the Vip2 pesticidal protein(SEQ ID NO:55), Isp2a from Brevibacillus laterosporus (SEQ ID NO:56) andIota toxin component Ia from Clostridium perfringens (SEQ ID NO:57). Thealignment shows the most highly conserved amino acid residueshighlighted in black, and highly conserved amino acid residueshighlighted in gray.

DETAILED DESCRIPTION

The present invention is drawn to compositions and methods forregulating pest resistance or tolerance in organisms, particularlyplants or plant cells. By “resistance” is intended that the pest (e.g.,insect) is killed upon ingestion or other contact with the polypeptidesof the invention. By “tolerance” is intended an impairment or reductionin the movement, feeding, reproduction, or other functions of the pest.The methods involve transforming organisms with a nucleotide sequenceencoding a pesticidal protein of the invention. In particular, thenucleotide sequences of the invention are useful for preparing plantsand microorganisms that possess pesticidal activity. Thus, transformedbacteria, plants, plant cells, plant tissues and seeds are provided.Compositions are pesticidal nucleic acids and proteins of Bacillus orother species. The sequences find use in the construction of expressionvectors for subsequent transformation into organisms of interest, asprobes for the isolation of other homologous (or partially homologous)genes, and for the generation of altered pesticidal proteins by methodsknown in the art, such as domain swapping or DNA shuffling. The proteinsfind use in controlling or killing lepidopteran, coleopteran, dipteran,and nematode pest populations and for producing compositions withpesticidal activity.

Plasmids containing the nucleotide sequences of the invention weredeposited in the permanent collection of the Agricultural ResearchService Culture Collection, Northern Regional Research Laboratory(NRRL), 1815 North University Street, Peoria, Ill. 61604, United Statesof America, in accordance with Table 1. This deposit will be maintainedunder the terms of the Budapest Treaty on the International Recognitionof the Deposit of Microorganisms for the Purposes of Patent Procedure.This deposit was made merely as a convenience for those of skill in theart and is not an admission that a deposit is required under 35 U.S.C. §112. TABLE 1 Microorganism Deposit NRRL Gene Strain Clone number DepositDate axmi-011 ATX13026 pAX4600 B-30961 Jul. 21, 2006 axmi-012 ATX13026pAX012 B-30955 Jul. 21, 2006 axmi-013 ATX13002 pAX013 B-30956 Jul. 21,2006 axmi-015 ATX13026 pAX015 B-30957 Jul. 21, 2006 axmi-019 ATX14875pAX019 B-30958 Jul. 21, 2006 axmi-044 ATX14759 pAX2599 B-30942 Jun. 15,2006 axmi-037 ATX1489 pAX2558 B-30939 Jun. 15, 2006 axmi-043 ATX15398pAX2597 B-30941 Jun. 15, 2006 axmi-033 ATX14833 pAX4341 B-50047 May 29,2007 axmi-034 ATX14833 pAX4341 B-50047 May 29, 2007 axmi-022 ATX13045pAX022 B-30959 Jul. 21, 2006 axmi-023 ATX13045 pAX023 B-30960 Jul. 21,2006 axmi-041 ATX21738 pAX4310 B-30943 Jun. 15, 2006 axmi-063 ATX12972pAX5036 B-50048 May 29, 2007 axmi-064 ATX12972 pAX5036 B-50048 May 29,2007

By “pesticidal toxin” or “pesticidal protein” is intended a toxin thathas toxic activity against one or more pests, including, but not limitedto, members of the Lepidoptera, Diptera, and Coleoptera orders, or theNematoda phylum, or a protein that has homology to such a protein.Pesticidal proteins have been isolated from organisms including, forexample, Bacillus sp., Clostridium bifermentans and Paenibacilluspopilliae. Pesticidal proteins include amino acid sequences deduced fromthe full-length nucleotide sequences disclosed herein, and amino acidsequences that are shorter than the full-length sequences, either due tothe use of an alternate downstream start site, or due to processing thatproduces a shorter protein having pesticidal activity. Processing mayoccur in the organism the protein is expressed in, or in the pest afteringestion of the protein.

Pesticidal proteins encompass delta-endotoxins. Delta-endotoxins includeproteins identified as cry1 through cry43, cyt1 and cyt2, and Cyt-liketoxin. There are currently over 250 known species of delta-endotoxinswith a wide range of specificities and toxicities. For an expansive listsee Crickmore et al. (1998), Microbiol. Mol. Biol. Rev. 62:807-813, andfor regular updates see Crickmore et al. (2003) “Bacillus thuringiensistoxin nomenclature,” atwww.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.

Also provided herein are nucleotide sequences encoding polypeptides withhomology to several other known classes of pesticidal protein toxins.For example, axmi-011, axmi-012, axmi-015, axmi-032, axmi-044, axmi-033,axmi-034, axmi-022, axmi-063, and axmi-064 demonstrate homology topesticidal binary toxins such as VIP, Bin, and MTX toxins. The VIP1/VIP2toxins (see, for example, U.S. Pat. No. 5,770,696, herein incorporatedby reference in its entirety) are binary pesticidal toxins that exhibitstrong activity on insects by a mechanism believed to involvereceptor-mediated endocytosis followed by cellular toxification, similarto the mode of action of other binary (“A/B”) toxins. A/B toxins such asVIP, C2, CDT, CST, or the B. anthracis edema and lethal toxins initiallyinteract with target cells via a specific, receptor-mediated binding of“B” components as monomers. These monomers then form homoheptamers. The“B” heptamer-receptor complex then acts as a docking platform thatsubsequently binds and allows the translocation of an enzymatic “A”component(s) into the cytosol via receptor-mediated endocytosis. Onceinside the cell's cytosol, “A” components inhibit normal cell functionby, for example, ADP-ribosylation of G-actin, or increasingintracellular levels of cyclic AMP (cAMP). See Barth et al. (2004)Microbiol Mol Biol Rev 68:373-402, herein incorporated by reference inits entirety.

Aside from the A/B type binary toxins, other types of binary toxins thatact as pesticidal proteins are known in the art. Cry34Ab1 and Cry35Ab1comprise a binary toxin with pesticidal activity that was identifiedfrom strain PS149B1 (Ellis et al. (2002) Appl Environ Microbiol.68:1137-45, herein incorporated by reference in its entirety). Thesetoxins have molecular masses of approximately 14 and 44 kDa,respectively. Other binary toxins with similar organization and homologyto Cry34Aa and Cry34Ab have been identified (Baum et al. (2004) ApplEnviron Microbiol. 70:4889-98, herein incorporated by reference in itsentirety).

BinA and BinB are proteins from Bacillus sphaericus that comprise amosquitocidal binary toxin protein (Baumann et al. (1991) Micriobiol.Rev. 55:425-36). Cry35 exhibits amino acid similarity to these BinA andBinB proteins. Cry36 (ET69) and Cry38 (ET75) (International PatentApplication No. WO/00/66742-B, herein incorporated by reference in itsentirety) are independently isolated peptides that also exhibit aminoacid similarity to BinA and BinB, and thus are likely to comprise binarytoxins.

Cry23 (also known as cryET33; U.S. Pat. No. 6,063,756, hereinincorporated by reference in its entirety) and Cry37 (also known ascryET34; U.S. Pat. No. 6,063,756, herein incorporated by reference inits entirety) also appear to be binary pesticidal toxins. Cry23 alsoexhibits homology to MTX2 and MTX3 toxins. The term “MTX” is used in theart to delineate a set of pesticidal proteins that are produced byBacillus sphaericus. The first of these, often referred to in the art asMTX1, is synthesized as a parasporal crystal which is toxic tomosquitoes. The major components of the crystal are two proteins of 51and 42 kDa, Since the presence of both proteins are required fortoxicity, MTX1 is considered a “binary” toxin (Baumann et al. (1991)Microbiol. Rev. 55:425-436).

By analysis of different Bacillus sphaericus strains with differingtoxicities, two new classes of MTX toxins have been identified. MTX2 andMTX3 represent separate, related classes of pesticidal toxins thatexhibit pesticidal activity. See, for example, Baumann et al. (1991)Microbiol. Rev. 55:425-436, herein incorporated by reference in itsentirety. MTX2 is a 100-kDa toxin. More recently MTX3 has beenidentified as a separate toxin, though the amino acid sequence of MTX3from B. sphaericus is 38% identical to the MTX2 toxin of B. sphaericusSSII-1 (Liu, et al. (1996) Appl. Environ. Microbiol. 62: 2174-2176).

Thus, provided herein are novel isolated nucleotide sequences thatconfer pesticidal activity. These isolated nucleotide sequences encodepolypeptides with homology to known delta-endotoxins or binary toxins.Also provided are the amino acid sequences of the pesticidal proteins.The protein resulting from translation of this gene allows cells tocontrol or kill pests that ingest it.

Isolated Nucleic Acid Molecules, and Variants and Fragments Thereof

One aspect of the invention pertains to isolated or recombinant nucleicacid molecules comprising nucleotide sequences encoding pesticidalproteins and polypeptides or biologically active portions thereof, aswell as nucleic acid molecules sufficient for use as hybridizationprobes to identify nucleic acid molecules encoding proteins with regionsof sequence homology. As used herein, the term “nucleic acid molecule”is intended to include DNA molecules (e.g., recombinant DNA, cDNA orgenomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

An “isolated” or “purified” nucleic acid molecule or protein, orbiologically active portion thereof, is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Preferably, an “isolated” nucleicacid is free of sequences (preferably protein encoding sequences) thatnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For purposes of the invention,“isolated” when used to refer to nucleic acid molecules excludesisolated chromosomes. For example, in various embodiments, the isolatednucleic acid molecule encoding a pesticidal protein can contain lessthan about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotidesequences that naturally flank the nucleic acid molecule in genomic DNAof the cell from which the nucleic acid is derived. A pesticidal proteinthat is substantially free of cellular material includes preparations ofprotein having less than about 30%, 20%, 10%, or 5% (by dry weight) ofnon-pesticidal protein (also referred to herein as a “contaminatingprotein”).

Nucleotide sequences encoding the proteins of the present inventioninclude the sequence set forth in SEQ ID NO:1, 3, 5, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 60, or the nucleotidesequence deposited in a bacterial host as Accession No. NRRL B-30961,B-30955, B-30956, B-30957, B-30958, B-30942, B-30939, B-30941, B-50047,B-50047, B-30959, B-30960, B-30943, B-50048, or B-50048, and variants,fragments, and complements thereof. By “complement” is intended anucleotide sequence that is sufficiently complementary to a givennucleotide sequence such that it can hybridize to the given nucleotidesequence to thereby form a stable duplex. The corresponding amino acidsequence for the pesticidal protein encoded by this nucleotide sequenceare set forth in SEQ ID NO:2, 4, 6, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37 or 61.

Nucleic acid molecules that are fragments of these nucleotide sequencesencoding pesticidal proteins are also encompassed by the presentinvention. By “fragment” is intended a portion of the nucleotidesequence encoding a pesticidal protein. A fragment of a nucleotidesequence may encode a biologically active portion of a pesticidalprotein, or it may be a fragment that can be used as a hybridizationprobe or PCR primer using methods disclosed below. Nucleic acidmolecules that are fragments of a nucleotide sequence encoding apesticidal protein comprise at least about 50, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 1100, 1200, 1300, 1350, 1400 contiguousnucleotides, or up to the number of nucleotides present in a full-lengthnucleotide sequence encoding a pesticidal protein disclosed herein (forexample, 957 nucleotides for SEQ ID NO:1; 927 nucleotides for SEQ IDNO:3, 1017 nucleotides for SEQ ID NO:5; 1422 nucleotides for SEQ IDNO:8, 1053 nucleotides for SEQ ID NO:10; 1062 nucleotides for SEQ IDNO:12, 942 nucleotides for SEQ ID NO:14, etc.) depending upon theintended use. By “contiguous” nucleotides is intended nucleotideresidues that are immediately adjacent to one another. Fragments of thenucleotide sequences of the present invention will encode proteinfragments that retain the biological activity of the pesticidal proteinand, hence, retain pesticidal activity. By “retains activity” isintended that the fragment will have at least about 30%, at least about50%, at least about 70%, 80%, 90%, 95% or higher of the pesticidalactivity of the pesticidal protein. Methods for measuring pesticidalactivity are well known in the art. See, for example, Czapla and Lang(1990) J. Econ. Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J.252:199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293;and U.S. Pat. No. 5,743,477, all of which are herein incorporated byreference in their entirety.

A fragment of a nucleotide sequence encoding a pesticidal protein thatencodes a biologically active portion of a protein of the invention willencode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250,300, 350, 400, 450 contiguous amino acids, or up to the total number ofamino acids present in a full-length pesticidal protein of the invention(for example, 318 amino acids for SEQ ID NO:2, 308 amino acids for SEQID NO:4, 338 amino acids for SEQ ID NO:6, 296 amino acids for SEQ IDNO:7, 473 amino acids for SEQ ID NO:9, 351 amino acids for SEQ ID NO:11,353 amino acids for SEQ ID NO:13, and 314 amino acids for SEQ ID NO:15,etc.).

Preferred pesticidal proteins of the present invention are encoded by anucleotide sequence sufficiently identical to the nucleotide sequence ofSEQ ID NO:1, 3, 5, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, or 60. By “sufficiently identical” is intended an amino acid ornucleotide sequence that has at least about 60% or 65% sequenceidentity, about 70% or 75% sequence identity, about 80% or 85% sequenceidentity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity compared to a reference sequence using one ofthe alignment programs described herein using standard parameters. Oneof skill in the art will recognize that these values can beappropriately adjusted to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning, and thelike.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. The percent identity between two sequences can bedetermined using techniques similar to those described below, with orwithout allowing gaps. In calculating percent identity, typically exactmatches are counted.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A nonlimiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTNand BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:403.BLAST nucleotide searches can be performed with the BLASTN program,score=100, wordlength=12, to obtain nucleotide sequences homologous topesticidal-like nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the BLASTX program, score=50,wordlength=3, to obtain amino acid sequences homologous to pesticidalprotein molecules of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-Blast can be used to perform an iterated search thatdetects distant relationships between molecules. See Altschul et al.(1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blastprograms, the default parameters of the respective programs (e.g.,BLASTX and BLASTN) can be used. Alignment may also be performed manuallyby inspection.

Another non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the ClustalW algorithm (Higgins et al.(1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences andaligns the entirety of the amino acid or DNA sequence, and thus canprovide data about the sequence conservation of the entire amino acidsequence. The ClustalW algorithm is used in several commerciallyavailable DNA/amino acid analysis software packages, such as the ALIGNXmodule of the Vector NTI Program Suite (Invitrogen Corporation,Carlsbad, Calif.). After alignment of amino acid sequences withClustalW, the percent amino acid identity can be assessed. Anon-limiting example of a software program useful for analysis ofClustalW alignments is GENEDOC™. GENEDOC™ (Karl Nicholas) allowsassessment of amino acid (or DNA) similarity and identity betweenmultiple proteins. Another non-limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller (1988) CABIOS 4:11-17. Such an algorithm isincorporated into the ALIGN program (version 2.0), which is part of theGCG Wisconsin Genetics Software Package, Version 10 (available fromAccelrys, Inc., 9685 Scranton Rd., San Diego, Calif., USA). Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12, and a gap penalty of 4can be used.

Unless otherwise stated, GAP Version 10, which uses the algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48(3):443-453, will be used todetermine sequence identity or similarity using the followingparameters: % identity and % similarity for a nucleotide sequence usingGAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoringmatrix; % identity or % similarity for an amino acid sequence using GAPweight of 8 and length weight of 2, and the BLOSUM62 scoring program.Equivalent programs may also be used. By “equivalent program” isintended any sequence comparison program that, for any two sequences inquestion, generates an alignment having identical nucleotide residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10.

The invention also encompasses variant nucleic acid molecules.“Variants” of the pesticidal protein encoding nucleotide sequencesinclude those sequences that encode the pesticidal proteins disclosedherein but that differ conservatively because of the degeneracy of thegenetic code as well as those that are sufficiently identical asdiscussed above. Naturally occurring allelic variants can be identifiedwith the use of well-known molecular biology techniques, such aspolymerase chain reaction (PCR) and hybridization techniques as outlinedbelow. Variant nucleotide sequences also include synthetically derivednucleotide sequences that have been generated, for example, by usingsite-directed mutagenesis but which still encode the pesticidal proteinsdisclosed in the present invention as discussed below. Variant proteinsencompassed by the present invention are biologically active, that isthey continue to possess the desired biological activity of the nativeprotein, that is, pesticidal activity. By “retains activity” is intendedthat the variant will have at least about 30%, at least about 50%, atleast about 70%, or at least about 80% of the pesticidal activity of thenative protein. Methods for measuring pesticidal activity are well knownin the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol.83: 2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marrone etal. (1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No.5,743,477, all of which are herein incorporated by reference in theirentirety.

The skilled artisan will further appreciate that changes can beintroduced by mutation of the nucleotide sequences of the inventionthereby leading to changes in the amino acid sequence of the encodedpesticidal proteins, without altering the biological activity of theproteins. Thus, variant isolated nucleic acid molecules can be createdby introducing one or more nucleotide substitutions, additions, ordeletions into the corresponding nucleotide sequence disclosed herein,such that one or more amino acid substitutions, additions or deletionsare introduced into the encoded protein. Mutations can be introduced bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Such variant nucleotide sequences are also encompassed bythe present invention.

For example, conservative amino acid substitutions may be made at one ormore, predicted, nonessential amino acid residues. A “nonessential”amino acid residue is a residue that can be altered from the wild-typesequence of a pesticidal protein without altering the biologicalactivity, whereas an “essential” amino acid residue is required forbiological activity. A “conservative amino acid substitution” is one inwhich the amino acid residue is replaced with an amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art. These families includeamino acids with basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

Delta-endotoxins generally have five conserved sequence domains, andthree conserved structural domains (see, for example, de Maagd et al.(2001) Trends Genetics 17:193-199). The first conserved structuraldomain consists of seven alpha helices and is involved in membraneinsertion and pore formation. Domain II consists of three beta-sheetsarranged in a Greek key configuration, and domain III consists of twoantiparallel beta-sheets in “jelly-roll” formation (de Maagd et al.,2001, supra). Domains II and III are involved in receptor recognitionand binding, and are therefore considered determinants of toxinspecificity.

Amino acid substitutions may be made in nonconserved regions that retainfunction. In general, such substitutions would not be made for conservedamino acid residues, or for amino acid residues residing within aconserved motif, where such residues are essential for protein activity.Examples of residues that are conserved and that may be essential forprotein activity include, for example, residues that are identicalbetween all proteins contained in an alignment of similar or relatedtoxins to the sequences of the invention (e.g., residues that areidentical between all proteins contained in the alignment in FIG. 1, 2,3, or 4). Examples of residues that are conserved but that may allowconservative amino acid substitutions and still retain activity include,for example, residues that have only conservative substitutions betweenall proteins contained in an alignment of similar or related toxins tothe sequences of the invention (e.g., residues that have onlyconservative substitutions between all proteins contained in thealignment in FIG. 1, 2, 3, or 4). However, one of skill in the art wouldunderstand that functional variants may have minor conserved ornonconserved alterations in the conserved residues.

Alternatively, variant nucleotide sequences can be made by introducingmutations randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forability to confer pesticidal activity to identify mutants that retainactivity. Following mutagenesis, the encoded protein can be expressedrecombinantly, and the activity of the protein can be determined usingstandard assay techniques.

Using methods such as PCR, hybridization, and the like correspondingpesticidal sequences can be identified, such sequences havingsubstantial identity to the sequences of the invention. See, forexample, Sambrook and Russell (2001) Molecular Cloning: A LaboratoryManual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)and Innis, et al. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, NY).

In a hybridization method, all or part of the pesticidal nucleotidesequence can be used to screen cDNA or genomic libraries. Methods forconstruction of such cDNA and genomic libraries are generally known inthe art and are disclosed in Sambrook and Russell, 2001, supra. Theso-called hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments, or other oligonucleotides, and may be labeledwith a detectable group such as ³²P, or any other detectable marker,such as other radioisotopes, a fluorescent compound, an enzyme, or anenzyme co-factor. Probes for hybridization can be made by labelingsynthetic oligonucleotides based on the known pesticidalprotein-encoding nucleotide sequence disclosed herein. Degenerateprimers designed on the basis of conserved nucleotides or amino acidresidues in the nucleotide sequence or encoded amino acid sequence canadditionally be used. The probe typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, at least about 25, at least about 50, 75, 100, 125, 150,175, or 200 consecutive nucleotides of nucleotide sequence encoding apesticidal protein of the invention or a fragment or variant thereof.Methods for the preparation of probes for hybridization are generallyknown in the art and are disclosed in Sambrook and Russell, 2001, supraherein incorporated by reference.

For example, an entire pesticidal protein sequence disclosed herein, orone or more portions thereof, may be used as a probe capable ofspecifically hybridizing to corresponding pesticidal protein-likesequences and messenger RNAs. To achieve specific hybridization under avariety of conditions, such probes include sequences that are unique andare preferably at least about 10 nucleotides in length, or at leastabout 20 nucleotides in length. Such probes may be used to amplifycorresponding pesticidal sequences from a chosen organism by PCR. Thistechnique may be used to isolate additional coding sequences from adesired organism or as a diagnostic assay to determine the presence ofcoding sequences in an organism. Hybridization techniques includehybridization screening of plated DNA libraries (either plaques orcolonies; see, for example, Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.).

Isolated Proteins and Variants and Fragments Thereof

Pesticidal proteins are also encompassed within the present invention.By “pesticidal protein” is intended a protein having the amino acidsequence set forth in SEQ ID NO:2, 4, 6, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37 or 61. Fragments, biologically activeportions, and variants thereof are also provided, and may be used topractice the methods of the present invention.

“Fragments” or “biologically active portions” include polypeptidefragments comprising amino acid sequences sufficiently identical to theamino acid sequence set forth in SEQ ID NO:2, 4, 6, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 61, and that exhibitpesticidal activity (for example, SEQ ID NO:7). A biologically activeportion of a pesticidal protein can be a polypeptide that is, forexample, 10, 25, 50, 100, 150, 200, 250 or more amino acids in length.Such biologically active portions can be prepared by recombinanttechniques and evaluated for pesticidal activity. Methods for measuringpesticidal activity are well known in the art. See, for example, Czaplaand Lang (1990) J. Econ. Entomol. 83:2480-2485; Andrews et al. (1988)Biochem. J. 252:199-206; Marrone et al. (1985) J. of Economic Entomology78:290-293; and U.S. Pat. No. 5,743,477, all of which are hereinincorporated by reference in their entirety. As used here, a fragmentcomprises at least 8 contiguous amino acids of SEQ ID NO:2, 4, 6, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 61. Theinvention encompasses other fragments, however, such as any fragment inthe protein greater than about 10, 20, 30, 50, 100, 150, 200, 250, or300 amino acids.

By “variants” is intended proteins or polypeptides having an amino acidsequence that is at least about 60%, 65%, about 70%, 75%, about 80%,85%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the amino acid sequence of SEQ ID NO:2, 4, 6, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 61. Variants also includepolypeptides encoded by a nucleic acid molecule that hybridizes to thenucleic acid molecule of SEQ ID NO:1, 3, 5, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, or 60, or a complement thereof, understringent conditions. Variants include polypeptides that differ in aminoacid sequence due to mutagenesis. Variant proteins encompassed by thepresent invention are biologically active, that is they continue topossess the desired biological activity of the native protein, that is,retaining pesticidal activity. Methods for measuring pesticidal activityare well known in the art. See, for example, Czapla and Lang (1990) J.Econ. Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J.252:199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293;and U.S. Pat. No. 5,743,477, all of which are herein incorporated byreference in their entirety.

Bacterial genes, such as the axmi genes of this invention, quite oftenpossess multiple methionine initiation codons in proximity to the startof the open reading frame. Often, translation initiation at one or moreof these start codons will lead to generation of a functional protein.These start codons can include ATG codons. However, bacteria such asBacillus sp. also recognize the codon GTG as a start codon, and proteinsthat initiate translation at GTG codons contain a methionine at thefirst amino acid. Furthermore, it is not often determined a priori whichof these codons are used naturally in the bacterium. Thus, it isunderstood that use of one of the alternate methionine codons may alsolead to generation of pesticidal proteins. These pesticidal proteins areencompassed in the present invention and may be used in the methods ofthe present invention.

Antibodies to the polypeptides of the present invention, or to variantsor fragments thereof, are also encompassed. Methods for producingantibodies are well known in the art (see, for example, Harlow and Lane(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.; U.S. Pat. No. 4,196,265).

Altered or Improved Variants

It is recognized that DNA sequences of a pesticidal protein may bealtered by various methods, and that these alterations may result in DNAsequences encoding proteins with amino acid sequences different thanthat encoded by a pesticidal protein of the present invention. Thisprotein may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions of one or moreamino acids of SEQ ID NO:2, 4, 6, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37 or 61, including up to about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10, about 15, about20, about 25, about 30, about 35, about 40, about 45, about 50, about55, about 60, about 65, about 70, about 75, about 80, about 85, about90, about 100, about 105, about 110, about 115, about 120, about 125,about 130, about 135, about 140, about 145, about 150, about 155, ormore amino acid substitutions, deletions or insertions. Methods for suchmanipulations are generally known in the art. For example, amino acidsequence variants of a pesticidal protein can be prepared by mutationsin the DNA. This may also be accomplished by one of several forms ofmutagenesis and/or in directed evolution. In some aspects, the changesencoded in the amino acid sequence will not substantially affect thefunction of the protein. Such variants will possess the desiredpesticidal activity. However, it is understood that the ability of apesticidal protein to confer pesticidal activity may be improved by theuse of such techniques upon the compositions of this invention. Forexample, one may express a pesticidal protein in host cells that exhibithigh rates of base misincorporation during DNA replication, such as XL-1Red (Stratagene, La Jolla, Calif.). After propagation in such strains,one can isolate the DNA (for example by preparing plasmid DNA, or byamplifying by PCR and cloning the resulting PCR fragment into a vector),culture the pesticidal protein mutations in a non-mutagenic strain, andidentify mutated genes with pesticidal activity, for example byperforming an assay to test for pesticidal activity. Generally, theprotein is mixed and used in feeding assays. See, for example Marrone etal. (1985) J. of Economic Entomology 78:290-293. Such assays can includecontacting plants with one or more pests and determining the plant'sability to survive and/or cause the death of the pests. Examples ofmutations that result in increased toxicity are found in Schnepf et al.(1998) Microbiol. Mol. Biol. Rev. 62:775-806.

Alternatively, alterations may be made to the protein sequence of manyproteins at the amino or carboxy terminus without substantiallyaffecting activity. This can include insertions, deletions, oralterations introduced by modern molecular methods, such as PCR,including PCR amplifications that alter or extend the protein codingsequence by virtue of inclusion of amino acid encoding sequences in theoligonucleotides utilized in the PCR amplification. Alternatively, theprotein sequences added can include entire protein-coding sequences,such as those used commonly in the art to generate protein fusions. Suchfusion proteins are often used to (1) increase expression of a proteinof interest (2) introduce a binding domain, enzymatic activity, orepitope to facilitate either protein purification, protein detection, orother experimental uses known in the art (3) target secretion ortranslation of a protein to a subcellular organelle, such as theperiplasmic space of Gram-negative bacteria, or the endoplasmicreticulum of eukaryotic cells, the latter of which often results inglycosylation of the protein.

Variant nucleotide and amino acid sequences of the present inventionalso encompass sequences derived from mutagenic and recombinogenicprocedures such as DNA shuffling. With such a procedure, one or moredifferent pesticidal protein coding regions can be used to create a newpesticidal protein possessing the desired properties. In this manner,libraries of recombinant polynucleotides are generated from a populationof related sequence polynucleotides comprising sequence regions thathave substantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between a pesticidal geneof the invention and other known pesticidal genes to obtain a new genecoding for a protein with an improved property of interest, such as anincreased insecticidal activity. Strategies for such DNA shuffling areknown in the art. See, for example, Stemmer (1994) Proc. Natl. Acad.Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri etal. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

Domain swapping or shuffling is another mechanism for generating alteredpesticidal proteins. Domains may be swapped between pesticidal proteins,resulting in hybrid or chimeric toxins with improved pesticidal activityor target spectrum. Methods for generating recombinant proteins andtesting them for pesticidal activity are well known in the art (see, forexample, Naimov et al. (2001) Appl. Environ. Microbiol. 67:5328-5330; deMaagd et al. (1996) Appl. Environ. Microbiol. 62:1537-1543; Ge et al.(1991) J. Biol. Chem. 266:17954-17958; Schnepf et al. (1990) J. Biol.Chem. 265:20923-20930; Rang et al. 91999) Appl. Environ. Microbiol.65:2918-2925).

Vectors

A pesticidal sequence of the invention may be provided in an expressioncassette for expression in a plant of interest. By “plant expressioncassette” is intended a DNA construct that is capable of resulting inthe expression of a protein from an open reading frame in a plant cell.Typically these contain a promoter and a coding sequence. Often, suchconstructs will also contain a 3′ untranslated region. Such constructsmay contain a “signal sequence” or “leader sequence” to facilitateco-translational or post-translational transport of the peptide tocertain intracellular structures such as the chloroplast (or otherplastid), endoplasmic reticulum, or Golgi apparatus.

By “signal sequence” is intended a sequence that is known or suspectedto result in cotranslational or post-translational peptide transportacross the cell membrane. In eukaryotes, this typically involvessecretion into the Golgi apparatus, with some resulting glycosylation.Insecticidal toxins of bacteria are often synthesized as protoxins,which are protolytically activated in the gut of the target pest (Chang(1987) Methods Enzymol. 153:507-516). In some embodiments of the presentinvention, the signal sequence is located in the native sequence, or maybe derived from a sequence of the invention. By “leader sequence” isintended any sequence that when translated, results in an amino acidsequence sufficient to trigger co-translational transport of the peptidechain to a subcellular organelle. Thus, this includes leader sequencestargeting transport and/or glycosylation by passage into the endoplasmicreticulum, passage to vacuoles, plastids including chloroplasts,mitochondria, and the like.

By “plant transformation vector” is intended a DNA molecule that isnecessary for efficient transformation of a plant cell. Such a moleculemay consist of one or more plant expression cassettes, and may beorganized into more than one “vector” DNA molecule. For example, binaryvectors are plant transformation vectors that utilize two non-contiguousDNA vectors to encode all requisite cis- and trans-acting functions fortransformation of plant cells (Hellens and Mullineaux (2000) Trends inPlant Science 5:446-451). “Vector” refers to a nucleic acid constructdesigned for transfer between different host cells. “Expression vector”refers to a vector that has the ability to incorporate, integrate andexpress heterologous DNA sequences or fragments in a foreign cell. Thecassette will include 5′ and 3′ regulatory sequences operably linked toa sequence of the invention. By “operably linked” is intended afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand, where necessary to join two protein coding regions, contiguous andin the same reading frame. The cassette may additionally contain atleast one additional gene to be cotransformed into the organism.Alternatively, the additional gene(s) can be provided on multipleexpression cassettes.

“Promoter” refers to a nucleic acid sequence that functions to directtranscription of a downstream coding sequence. The promoter togetherwith other transcriptional and translational regulatory nucleic acidsequences (also termed “control sequences”) are necessary for theexpression of a DNA sequence of interest.

Such an expression cassette is provided with a plurality of restrictionsites for insertion of the pesticidal sequence to be under thetranscriptional regulation of the regulatory regions.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a DNA sequence of the invention, and a translationaland transcriptional termination region (i.e., termination region)functional in plants. The promoter may be native or analogous, orforeign or heterologous, to the plant host and/or to the DNA sequence ofthe invention. Additionally, the promoter may be the natural sequence oralternatively a synthetic sequence. Where the promoter is “native” or“homologous” to the plant host, it is intended that the promoter isfound in the native plant into which the promoter is introduced. Wherethe promoter is “foreign” or “heterologous” to the DNA sequence of theinvention, it is intended that the promoter is not the native ornaturally occurring promoter for the operably linked DNA sequence of theinvention.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host, or may be derived from another source(i.e., foreign or heterologous to the promoter, the DNA sequence ofinterest, the plant host, or any combination thereof). Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; andJoshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

Where appropriate, the gene(s) may be optimized for increased expressionin the transformed host cell. That is, the genes can be synthesizedusing host cell-preferred codons for improved expression, or may besynthesized using codons at a host-preferred codon usage frequency.Generally, the GC content of the gene will be increased. See, forexample, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic AcidsRes. 17:477-498, herein incorporated by reference.

In one embodiment, the pesticidal protein is targeted to the chloroplastfor expression. In this manner, where the pesticidal protein is notdirectly inserted into the chloroplast, the expression cassette willadditionally contain a nucleic acid encoding a transit peptide to directthe pesticidal protein to the chloroplasts. Such transit peptides areknown in the art. See, for example, Von Heijne et al. (1991) Plant Mol.Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem.264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968;Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; andShah et al. (1986) Science 233:478-481.

The pesticidal gene to be targeted to the chloroplast may be optimizedfor expression in the chloroplast to account for differences in codonusage between the plant nucleus and this organelle. In this manner, thenucleic acids of interest may be synthesized using chloroplast-preferredcodons. See, for example, U.S. Pat. No. 5,380,831, herein incorporatedby reference.

Plant Transformation

Methods of the invention involve introducing a nucleotide construct intoa plant. By “introducing” is intended to present to the plant thenucleotide construct in such a manner that the construct gains access tothe interior of a cell of the plant. The methods of the invention do notrequire that a particular method for introducing a nucleotide constructto a plant is used, only that the nucleotide construct gains access tothe interior of at least one cell of the plant. Methods for introducingnucleotide constructs into plants are known in the art including, butnot limited to, stable transformation methods, transient transformationmethods, and virus-mediated methods.

By “plant” is intended whole plants, plant organs (e.g., leaves, stems,roots, etc.), seeds, plant cells, propagules, embryos and progeny of thesame. Plant cells can be differentiated or undifferentiated (e.g.callus, suspension culture cells, protoplasts, leaf cells, root cells,phloem cells, pollen).

“Transgenic plants” or “transformed plants” or “stably transformed”plants or cells or tissues refers to plants that have incorporated orintegrated exogenous nucleic acid sequences or DNA fragments into theplant cell. These nucleic acid sequences include those that areexogenous, or not present in the untransformed plant cell, as well asthose that may be endogenous, or present in the untransformed plantcell. “Heterologous” generally refers to the nucleic acid sequences thatare not endogenous to the cell or part of the native genome in whichthey are present, and have been added to the cell by infection,transfection, microinjection, electroporation, microprojection, or thelike.

Transformation of plant cells can be accomplished by one of severaltechniques known in the art. The pesticidal gene of the invention may bemodified to obtain or enhance expression in plant cells. Typically aconstruct that expresses such a protein would contain a promoter todrive transcription of the gene, as well as a 3′ untranslated region toallow transcription termination and polyadenylation. The organization ofsuch constructs is well known in the art. In some instances, it may beuseful to engineer the gene such that the resulting peptide is secreted,or otherwise targeted within the plant cell. For example, the gene canbe engineered to contain a signal peptide to facilitate transfer of thepeptide to the endoplasmic reticulum. It may also be preferable toengineer the plant expression cassette to contain an intron, such thatmRNA processing of the intron is required for expression.

Typically this “plant expression cassette” will be inserted into a“plant transformation vector”. This plant transformation vector may becomprised of one or more DNA vectors needed for achieving planttransformation. For example, it is a common practice in the art toutilize plant transformation vectors that are comprised of more than onecontiguous DNA segment. These vectors are often referred to in the artas “binary vectors”. Binary vectors as well as vectors with helperplasmids are most often used for Agrobacterium-mediated transformation,where the size and complexity of DNA segments needed to achieveefficient transformation is quite large, and it is advantageous toseparate functions onto separate DNA molecules. Binary vectors typicallycontain a plasmid vector that contains the cis-acting sequences requiredfor T-DNA transfer (such as left border and right border), a selectablemarker that is engineered to be capable of expression in a plant cell,and a “gene of interest” (a gene engineered to be capable of expressionin a plant cell for which generation of transgenic plants is desired).Also present on this plasmid vector are sequences required for bacterialreplication. The cis-acting sequences are arranged in a fashion to allowefficient transfer into plant cells and expression therein. For example,the selectable marker gene and the pesticidal gene are located betweenthe left and right borders. Often a second plasmid vector contains thetrans-acting factors that mediate T-DNA transfer from Agrobacterium toplant cells. This plasmid often contains the virulence functions (Virgenes) that allow infection of plant cells by Agrobacterium, andtransfer of DNA by cleavage at border sequences and vir-mediated DNAtransfer, as is understood in the art (Hellens and Mullineaux (2000)Trends in Plant Science 5:446-451). Several types of Agrobacteriumstrains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used forplant transformation. The second plasmid vector is not necessary fortransforming the plants by other methods such as microprojection,microinjection, electroporation, polyethylene glycol, etc.

In general, plant transformation methods involve transferringheterologous DNA into target plant cells (e.g. immature or matureembryos, suspension cultures, undifferentiated callus, protoplasts,etc.), followed by applying a maximum threshold level of appropriateselection (depending on the selectable marker gene) to recover thetransformed plant cells from a group of untransformed cell mass.Explants are typically transferred to a fresh supply of the same mediumand cultured routinely. Subsequently, the transformed cells aredifferentiated into shoots after placing on regeneration mediumsupplemented with a maximum threshold level of selecting agent. Theshoots are then transferred to a selective rooting medium for recoveringrooted shoot or plantlet. The transgenic plantlet then grows into amature plant and produces fertile seeds (e.g. Hiei et al. (1994) ThePlant Journal 6:271-282; Ishida et al. (1996) Nature Biotechnology14:745-750). Explants are typically transferred to a fresh supply of thesame medium and cultured routinely. A general description of thetechniques and methods for generating transgenic plants are found inAyres and Park (1994) Critical Reviews in Plant Science 13:219-239 andBommineni and Jauhar (1997) Maydica 42:107-120. Since the transformedmaterial contains many cells; both transformed and non-transformed cellsare present in any piece of subjected target callus or tissue or groupof cells. The ability to kill non-transformed cells and allowtransformed cells to proliferate results in transformed plant cultures.Often, the ability to remove non-transformed cells is a limitation torapid recovery of transformed plant cells and successful generation oftransgenic plants.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Generation oftransgenic plants may be performed by one of several methods, including,but not limited to, microinjection, electroporation, direct genetransfer, introduction of heterologous DNA by Agrobacterium into plantcells (Agrobacterium-mediated transformation), bombardment of plantcells with heterologous foreign DNA adhered to particles, ballisticparticle acceleration, aerosol beam transformation (U.S. PublishedApplication No. 20010026941; U.S. Pat. No. 4,945,050; InternationalPublication No. WO 91/00915; U.S. Published Application No. 2002015066),Lec1 transformation, and various other non-particle direct-mediatedmethods to transfer DNA.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530;Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab andMaliga (1993) EMBO J. 12:601-606. The method relies on particle gundelivery of DNA containing a selectable marker and targeting of the DNAto the plastid genome through homologous recombination. Additionally,plastid transformation can be accomplished by transactivation of asilent plastid-borne transgene by tissue-preferred expression of anuclear-encoded and plastid-directed RNA polymerase. Such a system hasbeen reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA91:7301-7305.

Following integration of heterologous foreign DNA into plant cells, onethen applies a maximum threshold level of appropriate selection in themedium to kill the untransformed cells and separate and proliferate theputatively transformed cells that survive from this selection treatmentby transferring regularly to a fresh medium. By continuous passage andchallenge with appropriate selection, one identifies and proliferatesthe cells that are transformed with the plasmid vector. Molecular andbiochemical methods can then be used to confirm the presence of theintegrated heterologous gene of interest into the genome of thetransgenic plant.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a nucleotide construct of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

Evaluation of Plant Transformation

Following introduction of heterologous foreign DNA into plant cells, thetransformation or integration of heterologous gene in the plant genomeis confirmed by various methods such as analysis of nucleic acids,proteins and metabolites associated with the integrated gene.

PCR analysis is a rapid method to screen transformed cells, tissue orshoots for the presence of incorporated gene at the earlier stage beforetransplanting into the soil (Sambrook and Russell (2001) MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). PCR is carried out using oligonucleotide primersspecific to the gene of interest or Agrobacterium vector background,etc.

Plant transformation may be confirmed by Southern blot analysis ofgenomic DNA (Sambrook and Russell, 2001, supra). In general, total DNAis extracted from the transformant, digested with appropriaterestriction enzymes, fractionated in an agarose gel and transferred to anitrocellulose or nylon membrane. The membrane or “blot” is then probedwith, for example, radiolabeled ³²P target DNA fragment to confirm theintegration of introduced gene into the plant genome according tostandard techniques (Sambrook and Russell, 2001, supra).

In Northern blot analysis, RNA is isolated from specific tissues oftransformant, fractionated in a formaldehyde agarose gel, and blottedonto a nylon filter according to standard procedures that are routinelyused in the art (Sambrook and Russell, 2001, supra). Expression of RNAencoded by the pesticidal gene is then tested by hybridizing the filterto a radioactive probe derived from a pesticidal gene, by methods knownin the art (Sambrook and Russell, 2001, supra).

Western blot, biochemical assays and the like may be carried out on thetransgenic plants to confirm the presence of protein encoded by thepesticidal gene by standard procedures (Sambrook and Russell, 2001,supra) using antibodies that bind to one or more epitopes present on thepesticidal protein.

Pesticidal Activity in Plants

In another aspect of the invention, one may generate transgenic plantsexpressing a pesticidal protein that has pesticidal activity. Methodsdescribed above by way of example may be utilized to generate transgenicplants, but the manner in which the transgenic plant cells are generatedis not critical to this invention. Methods known or described in the artsuch as Agrobacterium-mediated transformation, biolistic transformation,and non-particle-mediated methods may be used at the discretion of theexperimenter. Plants expressing a pesticidal protein may be isolated bycommon methods described in the art, for example by transformation ofcallus, selection of transformed callus, and regeneration of fertileplants from such transgenic callus. In such process, one may use anygene as a selectable marker so long as its expression in plant cellsconfers ability to identify or select for transformed cells.

A number of markers have been developed for use with plant cells, suchas resistance to chloramphenicol, the aminoglycoside G418, hygromycin,or the like. Other genes that encode a product involved in chloroplastmetabolism may also be used as selectable markers. For example, genesthat provide resistance to plant herbicides such as glyphosate,bromoxynil, or imidazolinone may find particular use. Such genes havebeen reported (Stalker et al. (1985) J. Biol. Chem. 263:6310-6314(bromoxynil resistance nitrilase gene); and Sathasivan et al. (1990)Nucl. Acids Res. 18:2188 (AHAS imidazolinone resistance gene).Additionally, the genes disclosed herein are useful as markers to assesstransformation of bacterial or plant cells. Methods for detecting thepresence of a transgene in a plant, plant organ (e.g., leaves, stems,roots, etc.), seed, plant cell, propagule, embryo or progeny of the sameare well known in the art. In one embodiment, the presence of thetransgene is detected by testing for pesticidal activity.

Fertile plants expressing a pesticidal protein may be tested forpesticidal activity, and the plants showing optimal activity selectedfor further breeding. Methods are available in the art to assay for pestactivity. Generally, the protein is mixed and used in feeding assays.See, for example Marrone et al. (1985) J. of Economic Entomology78:290-293.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplants of interest include, but are not limited to, corn (maize),sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton,rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape,Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato,cassaya, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana,avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond,oats, vegetables, ornamentals, and conifers.

Vegetables include, but are not limited to, tomatoes, lettuce, greenbeans, lima beans, peas, and members of the genus Curcumis such ascucumber, cantaloupe, and musk melon. Ornamentals include, but are notlimited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils,petunias, carnation, poinsettia, and chrysanthemum. Preferably, plantsof the present invention are crop plants (for example, maize, sorghum,wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice,soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape, etc.).

Use in Pesticidal Control

General methods for employing strains comprising a nucleotide sequenceof the present invention, or a variant thereof, in pesticide control orin engineering other organisms as pesticidal agents are known in theart. See, for example U.S. Pat. No. 5,039,523 and EP 0480762A2.

The Bacillus strains containing a nucleotide sequence of the presentinvention, or a variant thereof, or the microorganisms that have beengenetically altered to contain a pesticidal gene and protein may be usedfor protecting agricultural crops and products from pests. In one aspectof the invention, whole, i.e., unlysed, cells of a toxin(pesticide)-producing organism are treated with reagents that prolongthe activity of the toxin produced in the cell when the cell is appliedto the environment of target pest(s).

Alternatively, the pesticide is produced by introducing a pesticidalgene into a cellular host. Expression of the pesticidal gene results,directly or indirectly, in the intracellular production and maintenanceof the pesticide. In one aspect of this invention, these cells are thentreated under conditions that prolong the activity of the toxin producedin the cell when the cell is applied to the environment of targetpest(s). The resulting product retains the toxicity of the toxin. Thesenaturally encapsulated pesticides may then be formulated in accordancewith conventional techniques for application to the environment hostinga target pest, e.g., soil, water, and foliage of plants. See, forexample EPA 0192319, and the references cited therein. Alternatively,one may formulate the cells expressing a gene of this invention such asto allow application of the resulting material as a pesticide.

The active ingredients of the present invention are normally applied inthe form of compositions and can be applied to the crop area or plant tobe treated, simultaneously or in succession, with other compounds. Thesecompounds can be fertilizers, weed killers, cryoprotectants,surfactants, detergents, pesticidal soaps, dormant oils, polymers,and/or time-release or biodegradable carrier formulations that permitlong-term dosing of a target area following a single application of theformulation. They can also be selective herbicides, chemicalinsecticides, virucides, microbicides, amoebicides, pesticides,fungicides, bacteriocides, nematocides, molluscicides or mixtures ofseveral of these preparations, if desired, together with furtheragriculturally acceptable carriers, surfactants or application-promotingadjuvants customarily employed in the art of formulation. Suitablecarriers and adjuvants can be solid or liquid and correspond to thesubstances ordinarily employed in formulation technology, e.g. naturalor regenerated mineral substances, solvents, dispersants, wettingagents, tackifiers, binders or fertilizers. Likewise the formulationsmay be prepared into edible “baits” or fashioned into pest “traps” topermit feeding or ingestion by a target pest of the pesticidalformulation.

Methods of applying an active ingredient of the present invention or anagrochemical composition of the present invention that contains at leastone of the pesticidal proteins produced by the bacterial strains of thepresent invention include leaf application, seed coating and soilapplication. The number of applications and the rate of applicationdepend on the intensity of infestation by the corresponding pest.

The composition may be formulated as a powder, dust, pellet, granule,spray, emulsion, colloid, solution, or such like, and may be prepared bysuch conventional means as desiccation, lyophilization, homogenation,extraction, filtration, centrifugation, sedimentation, or concentrationof a culture of cells comprising the polypeptide. In all suchcompositions that contain at least one such pesticidal polypeptide, thepolypeptide may be present in a concentration of from about 1% to about99% by weight.

Lepidopteran, dipteran, or coleopteran pests may be killed or reduced innumbers in a given area by the methods of the invention, or may beprophylactically applied to an environmental area to prevent infestationby a susceptible pest. Preferably the pest ingests, or is contactedwith, a pesticidally-effective amount of the polypeptide. By“pesticidally-effective amount” is intended an amount of the pesticidethat is able to bring about death to at least one pest, or to noticeablyreduce pest growth, feeding, or normal physiological development. Thisamount will vary depending on such factors as, for example, the specifictarget pests to be controlled, the specific environment, location,plant, crop, or agricultural site to be treated, the environmentalconditions, and the method, rate, concentration, stability, and quantityof application of the pesticidally-effective polypeptide composition.The formulations may also vary with respect to climatic conditions,environmental considerations, and/or frequency of application and/orseverity of pest infestation.

The pesticide compositions described may be made by formulating eitherthe bacterial cell, crystal and/or spore suspension, or isolated proteincomponent with the desired agriculturally-acceptable carrier. Thecompositions may be formulated prior to administration in an appropriatemeans such as lyophilized, freeze-dried, desiccated, or in an aqueouscarrier, medium or suitable diluent, such as saline or other buffer. Theformulated compositions may be in the form of a dust or granularmaterial, or a suspension in oil (vegetable or mineral), or water oroil/water emulsions, or as a wettable powder, or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term “agriculturally-acceptable carrier” covers all adjuvants,inert components, dispersants, surfactants, tackifiers, binders, etc.that are ordinarily used in pesticide formulation technology; these arewell known to those skilled in pesticide formulation. The formulationsmay be mixed with one or more solid or liquid adjuvants and prepared byvarious means, e.g., by homogeneously mixing, blending and/or grindingthe pesticidal composition with suitable adjuvants using conventionalformulation techniques. Suitable formulations and application methodsare described in U.S. Pat. No. 6,468,523, herein incorporated byreference.

“Pest” includes but is not limited to, insects, fungi, bacteria,nematodes, mites, ticks, and the like. Insect pests include insectsselected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularlyColeoptera, Lepidoptera, and Diptera.

The order Coleoptera includes the suborders Adephaga and Polyphaga.Suborder Adephaga includes the superfamilies Caraboidea and Gyrinoidea,while suborder Polyphaga includes the superfamilies Hydrophiloidea,Staphylinoidea, Cantharoidea, Cleroidea, Elateroidea, Dascilloidea,Dryopoidea, Byrrhoidea, Cucujoidea, Meloidea, Mordelloidea,Tenebrionoidea, Bostrichoidea, Scarabaeoidea, Cerambycoidea,Chrysomeloidea, and Curculionoidea. Superfamily Caraboidea includes thefamilies Cicindelidae, Carabidae, and Dytiscidae. Superfamily Gyrinoideaincludes the family Gyrinidae. Superfamily Hydrophiloidea includes thefamily Hydrophilidae. Superfamily Staphylinoidea includes the familiesSilphidae and Staphylinidae. Superfamily Cantharoidea includes thefamilies Cantharidae and Lampyridae. Superfamily Cleroidea includes thefamilies Cleridae and Dermestidae. Superfamily Elateroidea includes thefamilies Elateridae and Buprestidae. Superfamily Cucujoidea includes thefamily Coccinellidae. Superfamily Meloidea includes the family Meloidae.Superfamily Tenebrionoidea includes the family Tenebrionidae.Superfamily Scarabaeoidea includes the families Passalidae andScarabaeidae. Superfamily Cerambycoidea includes the familyCerambycidae. Superfamily Chrysomeloidea includes the familyChrysomelidae. Superfamily Curculionoidea includes the familiesCurculionidae and Scolytidae.

The order Diptera includes the Suborders Nematocera, Brachycera, andCyclorrhapha. Suborder Nematocera includes the families Tipulidae,Psychodidae, Culicidae, Ceratopogonidae, Chironomidae, Simuliidae,Bibionidae, and Cecidomyiidae. Suborder Brachycera includes the familiesStratiomyidae, Tabanidae, Therevidae, Asilidae, Mydidae, Bombyliidae,and Dolichopodidae. Suborder Cyclorrhapha includes the Divisions Aschizaand Aschiza. Division Aschiza includes the families Phoridae, Syrphidae,and Conopidae. Division Aschiza includes the Sections Acalyptratae andCalyptratae. Section Acalyptratae includes the families Otitidae,Tephritidae, Agromyzidae, and Drosophilidae. Section Calyptrataeincludes the families Hippoboscidae, Oestridae, Tachinidae,Anthomyiidae, Muscidae, Calliphoridae, and Sarcophagidae.

The order Lepidoptera includes the families Papilionidae, Pieridae,Lycaenidae, Nymphalidae, Danaidae, Satyridae, Hesperiidae, Sphingidae,Saturniidae, Geometridae, Arctiidae, Noctuidae, Lymantriidae, Sesiidae,and Tineidae.

Insect pests of the invention for the major crops include: Maize:Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm;Helicoverpa zea, corn earworm; Spodoptera frugiperda, fall armyworm;Diatraea grandiosella, southwestern corn borer; Elasmopalpuslignosellus, lesser cornstalk borer; Diatraea saccharalis, surgarcaneborer; Diabrotica virgifera, western corn rootworm; Diabroticalongicornis barberi, northern corn rootworm; Diabrotica undecimpunctatahowardi, southern corn rootworm; Melanotus spp., wireworms; Cyclocephalaborealis, northern masked chafer (white grub); Cyclocephala immaculata,southern masked chafer (white grub); Popillia japonica, Japanese beetle;Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maizebillbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis,corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplusfemurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratorygrasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis,corn blot leafminer; Anaphothrips obscrurus, grass thrips; Solenopsismilesta, thief ant; Tetranychus urticae, twospotted spider mite;Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fallarmyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus,lesser cornstalk borer; Feltia subterranea, granulate cutworm;Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp.,wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria,corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphummaidis; corn leaf aphid; Sipha flava, yellow sugarcane aphid; Blissusleucopterus leucopterus, chinch bug; Contarinia sorghicola, sorghummidge; Tetranychus cinnabarinus, carmine spider mite; Tetranychusurticae, twospotted spider mite; Wheat: Pseudaletia unipunctata, armyworm; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus,lesser cornstalk borer; Agrotis orthogonia, western cutworm;Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus,cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabroticaundecimpunctata howardi, southern corn rootworm; Russian wheat aphid;Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid;Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Melanoplus sanguinipes,migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosismosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemyacoarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephuscinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower:Suleima helianthana, sunflower bud moth; Homoeosoma electellum,sunflower moth; zygogramma exclamationis, sunflower beetle; Bothyrusgibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seedmidge; Cotton: Heliothis virescens, cotton budworm; Helicoverpa zea,cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophoragossypiella, pink bollworm; Anthonomus grandis, boll weevil; Aphisgossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper;Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris,tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper;Melanoplus differentialis, differential grasshopper; Thrips tabaci,onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychuscinnabarinus, carmine spider mite; Tetranychus urticae, twospottedspider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodopterafrugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspisbrunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil;Sitophilus oryzae, rice weevil; Nephotettix nigropictus, riceleafhopper; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Soybean: Pseudoplusia includens, soybeanlooper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypenascabra, green cloverworm; Ostrinia nubilalis, European corn borer;Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm;Heliothis virescens, cotton budworm; Helicoverpa zea, cotton bollworm;Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peachaphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, greenstink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Hylemya platura, seedcornmaggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onionthrips; Tetranychus turkestani, strawberry spider mite; Tetranychusurticae, twospotted spider mite; Barley: Ostrinia nubilalis, Europeancorn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum,greenbug; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Euschistus servus, brown stink bug; Deliaplatura, seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobialatens, brown wheat mite; Oil Seed Rape: Brevicoryne brassicae, cabbageaphid; Phyllotreta cruciferae, Flea beetle; Mamestra configurata, Berthaarmyworm; Plutella xylostella, Diamond-back moth; Delia ssp., Rootmaggots.

Nematodes include parasitic nematodes such as root-knot, cyst, andlesion nematodes, including Heterodera spp., Meloidogyne spp., andGlobodera spp.; particularly members of the cyst nematodes, including,but not limited to, Heterodera glycines (soybean cyst nematode);Heterodera schachtii (beet cyst nematode); Heterodera avenae (cerealcyst nematode); and Globodera rostochiensis and Globodera pailida(potato cyst nematodes). Lesion nematodes include Pratylenchus spp.

Methods for Increasing Plant Yield

Methods for increasing plant yield are provided. The methods compriseintroducing into a plant or plant cell a polynucleotide comprising apesticidal sequence disclosed herein. As defined herein, the “yield” ofthe plant refers to the quality and/or quantity of biomass produced bythe plant. By “biomass” is intended any measured plant product. Anincrease in biomass production is any improvement in the yield of themeasured plant product. Increasing plant yield has several commercialapplications. For example, increasing plant leaf biomass may increasethe yield of leafy vegetables for human or animal consumption.Additionally, increasing leaf biomass can be used to increase productionof plant-derived pharmaceutical or industrial products. An increase inyield can comprise any statistically significant increase including, butnot limited to, at least a 1% increase, at least a 3% increase, at leasta 5% increase, at least a 10% increase, at least a 20% increase, atleast a 30%, at least a 50%, at least a 70%, at least a 100% or agreater increase in yield compared to a plant not expressing thepesticidal sequence.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Extraction of Plasmid DNA

Strains ATX14759, ATX14875, ATX13026, ATX13002, ATX9387, ATX13045,ATX21738, ATX14833, ATX1489, ATX15398 and ATX12972 were selected foranalysis. Pure cultures of each strain were grown in large quantities ofrich media. The cultures were centrifuged to harvest the cell pellet.The cell pellet was then prepared by treatment with SDS by methods knownin the art, resulting in breakage of the cell wall and release of DNA.Proteins and large genomic DNA were then precipitated by a high saltconcentration. The plasmid DNA was then precipitated with ethanol. Inseveral instances, the plasmid DNA was separated from any remainingchromosomal DNA by high-speed centrifugation through a cesium chloridegradient. Alternatively, the plasmid DNA was purified by binding to aresin, as known in the art. For each strain, the quality of the DNA waschecked by visualization on an agarose gel by methods known in the art.

Example 2 Cloning of Genes

DNA libraries were prepared from the plasmid DNA or each strain. Thismay be achieved in many ways as known in the art. For, example, thepurified plasmid DNA can be sheared into 5-10 kb sized fragments and the5′ and 3′ single stranded overhangs repaired using T4 DNA polymerase andKlenow fragment in the presence of all four dNTPs, as known in the art.Phosphates can then be attached to the 5′ ends by treatment with T4polynucleotide kinase, as known in the art. The repaired DNA fragmentscan then be ligated overnight into a standard high copy vector (i.e.pBLUESCRIPT™ SK+), suitably prepared to accept the inserts as known inthe art (for example by digestion with a restriction enzyme producingblunt ends).

The quality of the resulting DNA libraries was analyzed by digesting asubset of clones with a restriction enzyme known to have a cleavage siteflanking the cloning site. A high percentage of clones were determinedto contain inserts, ideally with an average insert size of 5-6 kb.

Example 3 High Throughput Sequencing of Library Plates

Once the DNA library quality was checked and confirmed, colonies weregrown in a rich broth in 2 ml 96-well blocks overnight at 37° C.,typically at a shaking speed of 350 rpm. The blocks were centrifuged toharvest the cells to the bottom of the block. The blocks were thenprepared by standard alkaline lysis prep in a high throughput format.

The end sequences of clones from this library were then determined for alarge number of clones from each block in the following manner: The DNAsequence of each clone chosen for analysis was determined using thefluorescent dye terminator sequencing technique (Applied Biosystems), bymethods known in the art using an automated DNA sequencing machine, andstandard oligonucleotide primers that anneal to the plasmid vector inthe region flanking the insert.

Example 4 Assembly and Screening of Sequencing Data

DNA sequences obtained were compiled into an assembly project andaligned together to form contigs. This can be done efficiently using acomputer program, such as Vector NTI, or alternatively by using thePhred/Phrap suite of DNA alignment and analysis programs. These contigs,along with any individual read that may not have been added to a contig,were compared to a compiled database of all classes of known pesticidalgenes. Contigs or individual reads identified as having identity to aknown endotoxin or pesticidal gene were analyzed further.

Example 5 axmi-037

From strain ATX1489, clone pAX2558 was found to contain an open readingframe with homology to “cry” type delta-endotoxins. This open readingframe was designated as axmi-037 (SEQ ID NO:16). Inspection of theaxmi-037 open reading frame suggests that more than one start codon maybe present. The two predicted start codons are the ATG codon beginningat nucleotide position 1 of SEQ ID NO:16, and a downstream ATG codon(represented by SEQ ID NO:18). The ATG at nucleotide 77 of SEQ ID NO:16has a ribosome binding site sequence (5′-G-G-A-G-G-3′), located atnucleotide positions 63-67 of SEQ ID NO:16. Based on the presence ofthis strong ribosome binding consensus sequence immediately upstream ofthis second start site, and the homology of the two predicted proteinsto other endotoxins, the translation product of the downstream startsite is herein designated AXMI-037 (SEQ ID NO:19). The longertranslation product, beginning at the ATG at nucleotide position 1 ofSEQ ID NO:16, is designated AXMI-37-2 (and set forth in SEQ ID NO:17).pAX2558 was deposited with the ARS Patent Strain Collection on Jun. 15,2006, and assigned NRRL B-30939. AXMI-37-2 exhibits 60% amino acididentity to the Cry7Aa1 endotoxin.

Example 6 axmi-019

From strain ATX14875, a clone was found to contain an open reading framewith homology to MTX family toxins. This open reading frame wasdesignated as axmi-019 (SEQ ID NO:10), and the encoded protein wasdesignated AXMI-019 (SEQ ID NO:11). By searching of public databases ofprotein sequences, such as the GenPept database, the C-terminal regionof AXMI-019 (starting at approximately amino acid 123 of SEQ ID NO:11)was found to have low homology to a class of toxins including Bacillusthuringiensis serovar darmstadiensis Cry14-4 toxin (SEQ ID NO:42;encoded by GENBANK® ID AAV70918.1), and the Bacillus sphaericus MTX2protein (SEQ ID NO:16, GENBANK® ID AAC44124.1).

Example 7 axmi-011, axmi-012 and axmi-015

From strain ATX13026, three individual clones were found to contain openreading frames with homology to MTX-like toxins. These open readingframes were designated axmi-011 (SEQ ID NO:1), axmi-012 (SEQ ID NO:3),and axmi-015 (SEQ ID NO:8), and the encoded proteins are designatedAXMI-011 (SEQ ID NO:2), AXMI-012 (SEQ ID NO:4), and AXMI-015 (SEQ IDNO:9), respectively. By searching of public databases of proteinsequences, AXMI-011 was found to have low homology to a class of toxinsincluding MTX2 (SEQ ID NO:16); AXMI-015 was found to have low homology(about 35% amino acid identify over 178 amino acids) to a mosquitocidaltoxin from Bacillus thuringiensis israelensis RBTH_(—)02046 (SEQ IDNO:41, GENBANK® ID gi/75761628:1-79; AXMI-012 was found to have homology(29% over 217 amino acids) to a class of toxins including the p42 binarytoxin of Bacillus sphaericus (SEQ ID NO:39; GENBANK® ID CAA73761).

Inspection of the axmi-011 coding region reveals the existence of analternate translational start site 12 nucleotides upstream of the ATGstart of axmi-011. This open reading frame contains a 5′ extension ofthe following twelve nucleotides 5′-G-T-G-A-T-G-A-A-A-A-A-A-3′ (SEQ IDNO:59)immediately upstream and adjacent to the axmi-11 open reading frame.This open reading frame is herein designated as axmi-011(long) (SEQ IDNO:60). Translation of axmi-011 utilizing the putative GTG start wouldresult in a modified AXMI-011 protein that contains an N-terminalextension of four amino acids (amino acid residues 1 through 4 of SEQ IDNO:61).

Analysis of the DNA context surrounding the two potential start sitesreveals a sequence with a good match to the consensus for a ribosomebinding site 5′ G-T-G-A-T-G-3′ (SEQ ID NO:62) positioned from −10 to −6nt relative to the ATG start codon of SEQ ID NO:1. This is a properposition for a bacterial ribosome binding site. No obvious homology tothe consensus ribosome start site is observed in the position 15 ntupstream of the putative GTG start site. Thus, the protein initiatedfrom the ATG start codon is designated AXMI-011 (SEQ ID NO:2). Theprotein encoded by translation initiated at the GTG start codon isdesignated AXMI-011(LONG) (SEQ ID NO:61).

Example 8 axmi-032

From strain ATX9387, a plasmid was found to contain an open readingframe with homology to pesticidal toxins. This open reading frame wasdesignated as axmi-032 (SEQ ID NO:12), and the encoded protein wasdesignated AXMI-032 (SEQ ID NO:13). By searching of public databases ofprotein sequences, such as the GenPept database, AXMI-032 was found tohave homology to a class of toxins including a presumed binary toxinfrom Bacillus thuringiensis (SEQ ID NO:43; GENBANK® Accession No.CAD30104.1) which is a possible two-domain toxin from Bacillusthuringiensis serovar israelensis.

Example 9 axmi-013

From strain ATX13002, a clone was found to contain an open reading framewith homology to “cry” type delta-endotoxins. This open reading framewas designated as axmi-013 (SEQ ID NO:5), and the encoded protein wasdesignated AXMI-013 (SEQ ID NO:6). By searching of public databases ofprotein sequences, the C-terminal region of AXMI-013 was found to have52% identity with the MTX3 toxin (SEQ ID NO:40: GENBANK® ID AAB36661).

Example 10 Expression of AXMI-013 in Bacillus

The insecticidal AXMI-013 gene is amplified by PCR and cloned into theBacillus expression vector pAX916 by methods well known in the art. Theresulting clone is assayed for expression of AXMI-013 protein aftertransformation into cells of a cry(−) Bacillus thuringiensis strain. ABacillus strain containing the axmi-013 clone and expressing theAXMI-013 insecticidal protein is grown in, for example, CYS media (10g/l Bacto-casitone; 3 g/l yeast extract; 6 g/l KH₂PO₄; 14 g/l K₂HPO₄;0.5 mM MgSO₄; 0.05 mM MnCl₂; 0.05 mM FeSO₄), until sporulation isevident by microscopic examination. Samples are prepared, and analyzedby polyacrylamide gel electrophoresis (PAGE). AXMI-013 is tested forinsecticidal activity in bioassays against important insect pests.

Inspection of the predicted amino acid sequence of AXMI-013 (SEQ IDNO:6) suggested that the N-terminus of the full-length AXMI-013 proteinmay comprise a signal peptide for secretion. The predicted site ofcleavage was estimated to be between the alanine at position 27 andlysine at position 28 of SEQ ID NO:6. Similarly, MTX3 (SEQ ID NO:40) ispredicted to possess a secretion signal peptide at its N-terminus (Liu,et al. (1996) Appl. Environ. Microbiol. 62:2174-2176).

The expressed AXMI-013 protein was excised from a polyacrylamide gel andsubjected to N-terminal sequence analysis as known in the art. TheN-terminal sequence identified by this analysis corresponded to aN-terminal truncation of the AXMI-013 protein, resulting in a truncatedpeptide with an N-terminus corresponding to the glutamine (Q) at aminoacid position 40 of SEQ ID NO:6. This truncated protein is referred toherein as AXMI-013(Q), and the amino acid sequence of this protein isprovided in SEQ ID NO:7. As known in the art, prediction of the exactsite of cleavage is somewhat difficult. Nonetheless, the cleavage atapproximately amino acid position 40 of SEQ ID NO:6 suggests that either(1) AXMI-013 is further processed after initial cleavage at amino acidpositions 27/28, or AXMI-013 has a novel secretion signal. In order toconfirm this, one skilled in the art may make gene fusion constructsutilizing (1) a heterologous protein and (2) using increasing lengthportions of AXMI-013. One can then test for secretion of the markerprotein and determine the processing sites by N-terminal sequencing.Other methods to determine the extent of the signal sequence are knownin the art.

Example 11 axmi-023 and axmi-041

From strain ATX13045, a plasmid clone was found to contain an openreading frame with homology to “cry” type delta-endotoxins. This openreading frame was designated as axmi-023 (SEQ ID NO:30), and the encodedprotein was designated AXMI-023 (SEQ ID NO:31). BLAST search of thenon-redundant ‘nr’ database demonstrates that AXMI-023 has low aminoacid identity (less than 30% amino acid identity) with the VIP2 proteintoxin, as well as several other presumed or known toxins. (GENBANK®Accession No. AA086513.1, SEQ ID NO:55)

From strain ATX21738, a plasmid clone was found to contain an openreading frame with homology to “cry” type delta-endotoxins. This openreading frame was designated as axmi-041 (SEQ ID NO:32), and the encodedprotein was designated AXMI-041 (SEQ ID NO:33). pAX4310 was depositedwith the ARS Patent Strain Collection on Jun. 15, 2006, and assignedNRRL B-30943. AXMI-041 is 21% identical to AXMI-023, and similarlyexhibits low amino acid identity (less than 30% amino acid identity)with the VIP2 protein toxin, as well as several other presumed or knowntoxins. A search of DNA and protein databases with the DNA sequences andamino acid sequences of AXMI-023 and AXMI-041 revealed that they arehomologous to known pesticidal proteins. FIG. 4 shows an alignment ofAXMI-023 with the Vip2 pesticidal protein (SEQ ID NO:55), and severalrelated toxins. AXMI-041 also shows homology with this class of toxins.

Example 12 AXMI-022 Defines a Novel Class of Pesticidal Proteins

From strain ATX13045, a plasmid clone was found to contain an openreading frame with homology to known insect toxins. This open readingframe was designated as axmi-022 (SEQ ID NO:28), and the encoded proteinwas designated AXMI-022 (SEQ ID NO:29).

The amino acid sequence of AXMI-022 is 64.9% identical to Vip1A(b) (SEQID NO:52; see also U.S. Pat. No. 5,770,696, herein incorporated byreference in its entirety) throughout the length of Vip1A(b), and hassignificant amino acid identity with several other binary proteintoxins. Further analysis of AXMI-022 revealed the following features ofthis polypeptide:

AXMI-022 is significantly longer than other binary proteins to which itshares homology, and encodes a peptide of 1,003 amino acids. Forexample, the Vip1A(b) protein is 834 amino acids in length;

Inspection of the DNA region surrounding the axmi-022 open reading frameshows no evidence for a second ORF encoding a toxin domain. The genesencoding binary toxins are typically physically closely linked. Mostoften, both the toxin and the receptor protein are organized as adjacentopen reading frames, and are often transcriptionally linked in anoperon.

The receptor binding region of AXMI-022 (from about amino acid 640 toabout amino acid 770 of SEQ ID NO:29) is different from other binarytoxins. The region of AXMI-022 corresponding to the region of binaryproteins involved in receptor binding is quite different in AXMI-022compared to other binary proteins. This is suggestive that AXMI-022binds to a different receptor than other binary proteins

The C-terminal 133 amino acids of AXMI-022 (starting about amino acid870 of SEQ ID NO:29) show amino acid homology to the Cry37Aa binaryprotein (SEQ ID NO:54; GENBANK® Accession No. AAF76376; U.S. Pat. No.6,063,756, herein incorporated by reference in its entirety). Thisregion has homology to Cry37Aa at a sequence identity of about 36%.Cry37Aa is in a different class of binary toxins than the Vip1-typetoxins. Cry37Aa belongs to the Cry34 family, which forms a binary toxinwith the Cry35 family. Cry34Ab1 is principally responsible for formingpores in lipid membranes, while Cry35Ab1 enhances the formation of pores(Masson, et al. (2004) Biochemistry 43:12349-57).

Thus, AXMI-022 appears to be a novel type of “single peptide” binarytoxin, having homology to multiple classes of binary proteins, with areceptor-binding component from one class of binary toxins directlyfused to a toxin component from a different class of binary toxins. Thisorganization of domains has not been previously predicted in the art.

Example 13 axmi-043

From strain ATX15398, pAX2597 was found to contain an open reading framewith homology to “cry” type delta-endotoxins. This open reading framewas designated as axmi-043. pAX2597 was deposited with the ARS PatentStrain Collection on Jun. 15, 2006, and assigned NRRL B-30941.Inspection of the axmi-043 open reading frame suggests that more thanone start codon may be present. The ATG at position 46 of SEQ ID NO:20has a ribosome binding site (5′ G-G-A-G-A-3′) (SEQ ID NO:63) starting atnucleotide 33 of SEQ ID NO:20. Based on the presence of this strongribosome binding consensus sequence immediately upstream of this secondstart site, and the homology of the two predicted proteins to otherendotoxins, we herein designate the translation product of the internalstart site (represented by SEQ ID NO:22) as AXMI-043 (SEQ ID NO:23), andthe longer protein as AXMI-043-2 (SEQ ID NO:21). AXMI-043 exhibits 93%amino acid identity to the AXMI-028 endotoxin, and AXMI-43-2 is 90%identical to AXMI-028 (SEQ ID NO:45 of this application, see also U.S.patent application Ser. No. 11/416,261, herein incorporated by referencein its entirety). AXMI-043 appears to be a “full-length endotoxin,” andcontains a C-terminal region (after the aspartic residue at position 629of the AXMI-043 amino acid sequence) often referred to in the art as anon-toxic domain or a “crystal domain.” AXMI-043 exhibits 54% amino acididentity with the Cry7Aa1 endotoxin (SEQ ID NO:46) throughout the fulllength of the protein.

Example 14 axmi-44

From strain ATX14759, pAX2599 was found to contain an open reading framewith homology to “cry” type delta-endotoxins. This open reading framewas designated as axmi-044 (SEQ ID NO:14), and the encoded protein wasdesignated AXMI-044 (SEQ ID NO:15). pAX2599 was deposited with the ARSPatent Strain Collection on Jun. 15, 2006, and assigned Accession No.NRRL B-30942. By searching of public databases of protein sequences,such as the GenPept database maintained by the NCBI (National Center forBiotechnology Information) AXMI-044 was found to have low homology tothe cry-15Aa/cry33 family of toxins (SEQ ID NO:44, GENBANK® ID 8928022),and to MTX2 (SEQ ID NO:38).

Example 15 axmi-033 and axmi-034

From strain ATX14833, a plasmid clone was found to contain two openreading frames with homology to insect toxins. The first open readingframe was designated as axmi-033 (SEQ ID NO:24), and the encoded proteinwas designated AXMI-033 (SEQ ID NO:25). AXMI-33 exhibits 61% amino acididentity to the 326 amino acid CryC35 insect toxin (SEQ ID NO:47,encoded by GENBANK® reference CAA63374). The second open reading framewas designated as axmi-034 (SEQ ID NO:26), and the encoded protein wasdesignated AXMI-034 (SEQ ID NO:27). AXMI-34 exhibits 45% amino acididentity with the CryC53 endotoxin. (SEQ ID NO:48, encoded by GENBANK®reference CAA67205).

axmi-033 and axmi-034 appear to comprise an operon. The ATG start ofaxmi-034 is immediately 3′ to, and in close proximity of (15 nucleotidesimmediately downstream of), the TAA stop codon of axmi-033. Thisorganization is well known in the art to suggest that two genes comprisean operon. Thus, the AXMI-033 and AXMI-034 proteins are likely to beco-expressed in their native strain. It is likely that the activities ofthe two proteins expressed together may be synergistic and superior tothe activity of the proteins expressed separately. pAX4341, a clonecontaining both axmi-033 and axmi-034 open reading frames, was depositedwith the ARS Patent Strain Collection on May 29, 2007, and assignedaccession number NRRL B-50047.

Example 16 axmi-063 and axmi-064

From strain ATX12972, a plasmid clone was found to contain two openreading frames with homology to insect toxins. The first open readingframe was designated as axmi-063 (SEQ ID NO:34), and the encoded proteinwas designated AXMI-063 (SEQ ID NO:35). AXMI-63 exhibits 53.1% aminoacid identity to the CryC35 insect toxin (SEQ ID NO:47, encoded byGENBANK® reference CAA63374). The second open reading frame wasdesignated as axmi-064 (SEQ ID NO:36), and the encoded protein wasdesignated AXMI-064 (SEQ ID NO:37). AXMI-64 exhibits 44.3% amino acididentity with the CryC53 endotoxin (SEQ ID NO:48, encoded by GENBANK®reference CAA67205).

axmi-063 and axmi-064 appear to comprise an operon. The ATG start ofaxmi-064 is immediately 3′ to, and in close proximity of, the TAA stopcodon of axmi-063. This is an organization well known in the art tosuggest that two genes comprise an operon. Thus, the AXMI-063 andAXMI-064 proteins are likely to be co-expressed in their native strain.It is likely that the activities of the two proteins expressed togethermay be synergistic and superior to the activity of the proteinsexpressed separately. pAX5036, a clone containing both axmi-063 andaxmi-064 open reading frames, was deposited with the ARS Patent StrainCollection on May 29, 2007, and assigned NRRL B-50048.

AXMI-033/AXMI-034 are similar to AXMI-063/AXMI-064. Analysis of theamino acid sequence of AXMI-033, AXMI-043, AXMI-063, and AXMI-064reveals that AXMI-033 and AXMI-063 share significant amino acididentity, and are 69% identical. Similarly AXMI-034 and AXMI-064 sharesignificant amino acid similarity (52% identical).

Example 17 Homology of AXMI-011, AXMI-012, AXMI-013, AXMI-015, AXMI-032,and AXMI-044 to Known Pesticidal Protein Genes

A search of protein databases with the amino acid sequences of theproteins of the invention reveal that they are homologous to knownpesticidal proteins. Comparison of the amino acid sequences of theproteins of the invention to the non-redundant (nr) database maintainedby the NCBI using the BLAST algorithm revealed the following proteins ashaving the strongest block of amino acid identity to the sequences ofthe invention (Table 2). Thus, the proteins of the invention are“pesticidal proteins.” TABLE 2 Amino Acid Identity of AXMI-011,AXMI-012, AXMI-013, AXMI-015, AXMI-032, and AXMI-044 to mosquito toxinsin public databases % Identity in PROTEIN Highest Blast Hit (nr) blockAXMI-011 MTX2 28% AXMI-012 P42 component of binary toxin 29% AXMI-013MTX3 52% AXMI-015 RBTH_02046 35% AXMI-019 Cry14-4, MTX2 35%, 30%AXMI-032 GENBANK ® ID CAD30104.1 19% AXMI-044 cry15Aa, MTX2 30%, 30%

Example 18 Additional Assays for Pesticidal Activity

The ability of a pesticidal protein to act as a pesticide upon a pest isoften assessed in a number of ways. One way well known in the art is toperform a feeding assay. In such a feeding assay, one exposes the pestto a sample containing either compounds to be tested, or controlsamples. Often this is performed by placing the material to be tested,or a suitable dilution of such material, onto a material that the pestwill ingest, such as an artificial diet. The material to be tested maybe composed of a liquid, solid, or slurry. The material to be tested maybe placed upon the surface and then allowed to dry. Alternatively, thematerial to be tested may be mixed with a molten artificial diet, thendispensed into the assay chamber. The assay chamber may be, for example,a cup, a dish, or a well of a microtiter plate.

Assays for sucking pests (for example aphids) may involve separating thetest material from the insect by a partition, ideally a portion that canbe pierced by the sucking mouth parts of the sucking insect, to allowingestion of the test material. Often the test material is mixed with afeeding stimulant, such as sucrose, to promote ingestion of the testcompound.

Other types of assays can include microinjection of the test materialinto the mouth, or gut of the pest, as well as development of transgenicplants, followed by test of the ability of the pest to feed upon thetransgenic plant. Plant testing may involve isolation of the plant partsnormally consumed, for example, small cages attached to a leaf, orisolation of entire plants in cages containing insects.

Other methods and approaches to assay pests are known in the art, andcan be found, for example in Robertson and Preisler, eds. (1992)Pesticide bioassays with arthropods, CRC, Boca Raton, Fla.Alternatively, assays are commonly described in the journals ArthropodManagement Tests and Journal of Economic Entomology or by discussionwith members of the Entomological Society of America (ESA).

Example 19 Vectoring of axmi Genes for Plant Expression

The coding regions of the invention are connected with appropriatepromoter and terminator sequences for expression in plants. Suchsequences are well known in the art and may include the rice actinpromoter or maize ubiquitin promoter for expression in monocots, theArabidopsis UBQ3 promoter or CaMV 35S promoter for expression in dicots,and the nos or PinII terminators. Techniques for producing andconfirming promoter-gene-terminator constructs also are well known inthe art. In one aspect of the invention, synthetic DNA sequences aredesigned and generated. These synthetic sequences have alterednucleotide sequence relative to the parent sequence, but encode proteinsthat are essentially identical to the parent AXMI protein.

In another aspect of the invention, modified versions of the syntheticgenes are designed such that the resulting peptide is targeted to aplant organelle, such as the endoplasmic reticulum or the apoplast.Peptide sequences known to result in targeting of fusion proteins toplant organelles are known in the art. For example, the N-terminalregion of the acid phosphatase gene from the White Lupin Lupinus albus(GENEBANK® ID GI: 14276838, Miller et al. (2001) Plant Physiology 127:594-606) is known in the art to result in endoplasmic reticulumtargeting of heterologous proteins. If the resulting fusion protein alsocontains an endoplasmic reticulum retention sequence comprising thepeptide N-terminus-lysine-aspartic acid-glutamic acid-leucine (i.e., the“KDEL” motif (SEQ ID NO:58)) at the C-terminus, the fusion protein willbe targeted to the endoplasmic reticulum. If the fusion protein lacks anendoplasmic reticulum targeting sequence at the C-terminus, the proteinwill be targeted to the endoplasmic reticulum, but will ultimately besequestered in the apoplast.

Thus, this gene encodes a fusion protein that contains the N-terminalthirty-one amino acids of the acid phosphatase gene from the White LupinLupinus albus (GENBANK® ID GI: 14276838, Miller et al., 2001, supra)fused to the N-terminus of the AXMI sequence, as well as the KDELsequence at the C-terminus. Thus, the resulting protein is predicted tobe targeted the plant endoplasmic reticulum upon expression in a plantcell.

The plant expression cassettes described above are combined with anappropriate plant selectable marker to aid in the selection oftransformed cells and tissues, and ligated into plant transformationvectors. These may include binary vectors from Agrobacterium-mediatedtransformation or simple plasmid vectors for aerosol or biolistictransformation.

Example 20 Vectoring of axmi Genes for Plant Expression

The coding region DNA of the axmi genes of the invention are operablyconnected with appropriate promoter and terminator sequences forexpression in plants. Such sequences are well known in the art and mayinclude the rice actin promoter or maize ubiquitin promoter forexpression in monocots, the Arabidopsis UBQ3 promoter or CaMV 35Spromoter for expression in dicots, and the nos or PinII terminators.Techniques for producing and confirming promoter-gene-terminatorconstructs also are well known in the art.

The plant expression cassettes described above are combined with anappropriate plant selectable marker to aid in the selections oftransformed cells and tissues, and ligated into plant transformationvectors. These may include binary vectors from Agrobacterium-mediatedtransformation or simple plasmid vectors for aerosol or biolistictransformation.

Example 21 Transformation of Maize Cells with the Pesticidal ProteinGenes Described Herein

Maize ears are best collected 8-12 days after pollination. Embryos areisolated from the ears, and those embryos 0.8-1.5 mm in size arepreferred for use in transformation. Embryos are plated scutellumside-up on a suitable incubation media, such as DN62A5S media (3.98 g/LN6 Salts; 1 mL/L (of 1000× Stock) N6 Vitamins; 800 mg/L L-Asparagine;100 mg/L Myo-inositol; 1.4 g/L L-Proline; 100 mg/L Casamino acids; 50g/L sucrose; 1 mL/L (of 1 mg/mL Stock) 2,4-D). However, media and saltsother than DN62A5S are suitable and are known in the art. Embryos areincubated overnight at 25° C. in the dark. However, it is not necessaryper se to incubate the embryos overnight.

The resulting explants are transferred to mesh squares (30-40 perplate), transferred onto osmotic media for about 30-45 minutes, thentransferred to a beaming plate (see, for example, PCT Publication No.WO/0138514 and U.S. Pat. No. 5,240,842).

DNA constructs designed to the genes of the invention in plant cells areaccelerated into plant tissue using an aerosol beam accelerator, usingconditions essentially as described in PCT Publication No. WO/0138514.After beaming, embryos are incubated for about 30 min on osmotic media,and placed onto incubation media overnight at 25° C. in the dark. Toavoid unduly damaging beamed explants, they are incubated for at least24 hours prior to transfer to recovery media. Embryos are then spreadonto recovery period media, for about 5 days, 25° C. in the dark, thentransferred to a selection media. Explants are incubated in selectionmedia for up to eight weeks, depending on the nature and characteristicsof the particular selection utilized. After the selection period, theresulting callus is transferred to embryo maturation media, until theformation of mature somatic embryos is observed. The resulting maturesomatic embryos are then placed under low light, and the process ofregeneration is initiated by methods known in the art. The resultingshoots are allowed to root on rooting media, and the resulting plantsare transferred to nursery pots and propagated as transgenic plants.Materials DN62A5S Media Components Per Liter Source Chu's N6 Basal 3.98g/L Phytotechnology Labs Salt Mixture (Prod. No. C 416) Chu's N6 Vitamin1 mL/L (of Phytotechnology Labs Solution 1000× Stock) (Prod. No. C 149)L-Asparagine 800 mg/L Phytotechnology Labs Myo-inositol 100 mg/L SigmaL-Proline 1.4 g/L Phytotechnology Labs Casamino acids 100 mg/L FisherScientific Sucrose 50 g/L Phytotechnology Labs 2,4-D (Prod. 1 mL/L (ofSigma No. D-7299) 1 mg/mL Stock)

The pH of the solution is adjusted to pH 5.8 with 1N KOH/1N KCl, Gelrite(Sigma) is added at a concentration up to 3 g/L, and the media isautoclaved. After cooling to 50° C., 2 ml/L of a 5 mg/ml stock solutionof silver nitrate (Phytotechnology Labs) is added.

Example 22 Transformation of the Pesticidal Genes of the Invention inPlant Cells by Agrobacterium-Mediated Transformation

Ears are best collected 8-12 days after pollination. Embryos areisolated from the ears, and those embryos 0.8-1.5 mm in size arepreferred for use in transformation. Embryos are plated scutellumside-up on a suitable incubation media, and incubated overnight at 25°C. in the dark. However, it is not necessary per se to incubate theembryos overnight. Embryos are contacted with an Agrobacterium straincontaining the appropriate vectors for Ti plasmid mediated transfer forabout 5-10 min, and then plated onto co-cultivation media for about 3days (25° C. in the dark). After co-cultivation, explants aretransferred to recovery period media for about five days (at 25° C. inthe dark). Explants are incubated in selection media for up to eightweeks, depending on the nature and characteristics of the particularselection utilized. After the selection period, the resulting callus istransferred to embryo maturation media, until the formation of maturesomatic embryos is observed. The resulting mature somatic embryos arethen placed under low light, and the process of regeneration isinitiated as known in the art.

Example 23 Soil Infestation of Plants Expressing a Gene of the Inventionwith Western Corn Rootworm

Transgenic plants containing an axmi gene of the invention under thecontrol of a plant promoter are tested for resistance to infestation byWestern corn rootworm (WCRW). Plantlets are transplanted from tissueculture media to root trainer (clamshell) pots known in the art to beuseful for growth of plantlets in soil. Plants are grown for about 2weeks in a greeenhouse. Transgenic plants, as well as untransformedcontrols, are infested with approximately 1,000 WCRW eggs. WCRW eggs arepreincubated such that eggs are at the point of hatching when infestedonto the plants. Plants are held for about four weeks, or until controlsexhibited obvious damage due to the rootworms. At this stage, plants arepulled from pots, roots are washed, and damage evaluated. Severalindependent events are examined for reduced damage from WCRW infestationrelative to non-transformed control plants.

The resulting shoots are allowed to root on rooting media, and theresulting plants are transferred to nursery pots and propagated astransgenic plants.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. An isolated nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of: a) the nucleotide sequence of SEQID NO:18, 1, 3, 5, 8, 10, 12, 14, 16, 20, 22, 24, 26, 28, 30, 32, 34,36, or 60, or a complement thereof; b) a nucleotide sequence having atleast 80% sequence identity to the nucleotide sequence of SEQ ID NO:18,1, 3, 5, 8, 10, 12, 14, 16, 20, 24, 26, 28, 30, 32, 34, 36, or 60, or acomplement thereof; c) a nucleotide sequence having at least 95%sequence identity to the nucleotide sequence of SEQ ID NO:22, or acomplement thereof; d) a nucleotide sequence that encodes a polypeptidecomprising the amino acid sequence of SEQ ID NO:19, 2, 4, 6, 7, 9, 11,13, 15, 17, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 61; e) a nucleotidesequence that encodes a polypeptide comprising an amino acid sequencehaving at least 80% sequence identity to the amino acid sequence of SEQID NO:19, 2, 4, 6, 7, 9, 11, 13, 15, 17, 21, 25, 27, 29, 31, 33, 35, 37or 61; f) a nucleotide sequence that encodes a polypeptide comprising anamino acid sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO:23; and, g) the nucleotide sequence of theDNA insert of the plasmid deposited as Accession No. NRRL B-30961,B-30955, B-30956, B-30957, B-30958, B-30942, B-30939, B-30941, B-50047,B-50047, B-30959, B-30960, B-30943, B-50048, or B-50048, or a complementthereof.
 2. The isolated nucleic acid molecule of claim 1, wherein saidnucleotide sequence is a synthetic sequence that has been designed forexpression in a plant.
 3. A vector comprising the nucleic acid moleculeof claim
 1. 4. The vector of claim 3, further comprising a nucleic acidmolecule encoding a heterologous polypeptide.
 5. A host cell thatcontains the vector of claim
 3. 6. The host cell of claim 5 that is abacterial host cell.
 7. The host cell of claim 5 that is a plant cell.8. A transgenic plant comprising the host cell of claim
 7. 9. Thetransgenic plant of claim 8, wherein said plant is selected from thegroup consisting of maize, sorghum, wheat, cabbage, sunflower, tomato,crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane,tobacco, barley, and oilseed rape.
 10. A transgenic seed comprising thenucleic acid molecule of claim
 1. 11. An isolated polypeptide withpesticidal activity, selected from the group consisting of: a) apolypeptide comprising the amino acid sequence of SEQ ID NO:19, 2, 4, 6,7, 9, 11, 13, 15, 17, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 61; b) apolypeptide comprising an amino acid sequence having at least 80%sequence identity to the amino acid sequence of SEQ ID NO:19, 2, 4, 6,7, 9, 11, 13, 15, 17, 21, 25, 27, 29, 31, 33, 35, 37 or 61; c) apolypeptide comprising an amino acid sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO:23; d) apolypeptide that is encoded by the nucleotide sequence of SEQ ID NO:18,1, 3, 5, 8, 10, 12, 14, 16, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 60;e) a polypeptide that is encoded by a nucleotide sequence that is atleast 80% identical to the nucleotide sequence of SEQ ID NO:18, 1, 3, 5,8, 10, 12, 14, 16, 20, 24, 26, 28, 30, 32, 34, 36, or 60; f) apolypeptide that is encoded by a nucleotide sequence that is at least95% identical to the nucleotide sequence of SEQ ID NO:22; and, g) apolypeptide encoded by the nucleotide sequence of the DNA insert of theplasmid deposited as Accession No. NRRL B-30961, B-30955, B-30956,B-30957, B-30958, B-30942, B-30939, B-30941, B-50047, B-50047, B-30959,B-30960, B-30943, B-50048, or B-50048.
 12. The polypeptide of claim 11further comprising heterologous amino acid sequences.
 13. A compositioncomprising the polypeptide of claim
 11. 14. The composition of claim 13,wherein said composition is selected from the group consisting of apowder, dust, pellet, granule, spray, emulsion, colloid, and solution.15. The composition of claim 13, wherein said composition is prepared bydesiccation, lyophilization, homogenization, extraction, filtration,centrifugation, sedimentation, or concentration of a culture ofbacterial cells.
 16. The composition of claim 13, comprising from about1% to about 99% by weight of said polypeptide.
 17. A method forcontrolling a lepidopteran, coleopteran, nematode, or dipteran pestpopulation comprising contacting said population with apesticidally-effective amount of a polypeptide of claim
 11. 18. A methodfor killing a lepidopteran, coleopteran, nematode, or dipteran pest,comprising contacting said pest with, or feeding to said pest, apesticidally-effective amount of a polypeptide of claim
 11. 19. A methodfor producing a polypeptide with pesticidal activity, comprisingculturing the host cell of claim 4 under conditions in which the nucleicacid molecule encoding the polypeptide is expressed.
 20. A plant havingstably incorporated into its genome a DNA construct comprising anucleotide sequence that encodes a protein having pesticidal activity,wherein said nucleotide sequence is selected from the group consistingof: a) the nucleotide sequence of SEQ ID NO:18, 1, 3, 5, 8, 10, 12, 14,16, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 60, or a complement thereof;b) a nucleotide sequence having at least 80% sequence identity to thenucleotide sequence of SEQ ID NO:18, 1, 3, 5, 8, 10, 12, 14, 16, 20, 24,26, 28, 30, 32, 34, 36, or 60, or a complement thereof; c) a nucleotidesequence having at least 95% sequence identity to the nucleotidesequence of SEQ ID NO:22, or a complement thereof; d) a nucleotidesequence that encodes a polypeptide comprising the amino acid sequenceof SEQ ID NO:19, 2, 4, 6, 7, 9, 11, 13, 15, 17, 21, 23, 25, 27, 29, 31,33, 35, 37 or 61; e) a nucleotide sequence that encodes a polypeptidecomprising an amino acid sequence having at least 80% sequence identityto the amino acid sequence of SEQ ID NO:19, 2, 4, 6, 7, 9, 11, 13, 15,17, 21, 25, 27, 29, 31, 33, 35, 37 or 61; f) a nucleotide sequence thatencodes a polypeptide comprising an amino acid sequence having at least95% sequence identity to the amino acid sequence of SEQ ID NO:23; and,g) the nucleotide sequence of the DNA insert of the plasmid deposited asAccession No. NRRL B-30961, B-30955, B-30956, B-30957, B-30958, B-30942,B-30939, B-30941, B-50047, B-50047, B-30959, B-30960, B-30943, B-50048,or B-50048, or a complement thereof; wherein said nucleotide sequence isoperably linked to a promoter that drives expression of a codingsequence in a plant cell.
 21. The plant of claim 20, wherein said plantis a plant cell.
 22. A method for protecting a plant from a pest,comprising introducing into said plant or cell thereof at least oneexpression vector comprising a nucleotide sequence that encodes apesticidal polypeptide, wherein said nucleotide sequence is selectedfrom the group consisting of: a) the nucleotide sequence of SEQ IDNO:18, 1, 3, 5, 8, 10, 12, 14, 16, 20, 22, 24, 26, 28, 30, 32, 34, 36,or 60, or a complement thereof; b) a nucleotide sequence having at least80% sequence identity to the nucleotide sequence of SEQ ID NO:18, 1, 3,5, 8, 10, 12, 14, 16, 20, 24, 26, 28, 30, 32, 34, 36, or 60, or acomplement thereof; c) a nucleotide sequence having at least 95%sequence identity to the nucleotide sequence of SEQ ID NO:22, or acomplement thereof; d) a nucleotide sequence that encodes a polypeptidecomprising the amino acid sequence of SEQ ID NO:19, 2, 4, 6, 7, 9, 11,13, 15, 17, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 61; e) a nucleotidesequence that encodes a polypeptide comprising an amino acid sequencehaving at least 80% sequence identity to the amino acid sequence of SEQID NO:19, 2, 4, 6, 7, 9, 11, 13, 15, 17, 21, 25, 27, 29, 31, 33, 35, 37or 61; f) a nucleotide sequence that encodes a polypeptide comprising anamino acid sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO:23; and, g) the nucleotide sequence of theDNA insert of the plasmid deposited as Accession No. NRRL B-30961,B-30955, B-30956, B-30957, B-30958, B-30942, B-30939, B-30941, B-50047,B-50047, B-30959, B-30960, B-30943, B-50048, or B-50048, or a complementthereof.
 23. The method of claim 22, wherein said plant produces apesticidal polypeptide having pesticidal activity against alepidopteran, coleopteran, nematode, or dipteran pest.